Method of treating neurodegenerative disease

ABSTRACT

Aspects featured in the invention relate to compositions and methods for inhibiting alpha-synuclein (SNCA) gene expression, such as for the treatment of neurodegenerative disorders. An anti-SNCA agent featured herein that targets the SNCA gene can have been modified to alter distribution in favor of neural cells.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/991,286, filed Nov. 17, 2004, which is a continuation-in-part ofInternational Application No. PCT/US2004/018271, filed Jun. 9, 2004,which claims the benefit of U.S. Provisional Application No. 60/476,947,filed Jun. 9, 2003. The contents of these applications are incorporatedherein by reference in their entirety.

GOVERNMENT SUPPORT

The work described herein was carried out, at least in part, using fundsfrom the U.S. government under grant number ES10751 awarded by NationalInstitute of Environmental Health Sciences, and grant numbers NS33978and NS40256 awarded by the National Institute of Neurological Disordersand Stroke. The government may therefore have certain rights in theinvention.

TECHNICAL FIELD

This invention relates to methods and compositions for treatingneurodegenerative disease, and more particularly to the downregulationof the alpha-synuclein gene for the treatment of synucleinopathies.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391:806-811, 1998). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function.

Expression of the SNCA gene produces the protein alpha-synuclein.Mutations in the SNCA gene and SNCA gene multiplications have beenlinked to familial Parkinson's disease (PD). PD patients demonstratealpha-synuclein protein aggregates in the brain. Similar aggregates areobserved in patients diagnosed with sporadic PD, Alzheimer's Disease,multiple system atrophy, and Lewy body dementia.

SUMMARY

Aspects of the invention relate to compositions for inhibitingalpha-synuclein (SNCA) expression, and methods of using thosecompositions. In one aspect, the invention features a method of treatinga subject by administering an agent which inhibits expression of SNCA.In a preferred embodiment, the subject is a mammal, such as a human,e.g., a subject diagnosed as having, or at risk for developing, aneurodegenerative disorder. The inhibition can be effected at any level,e.g., at the level of transcription, the level of translation, orpost-transitionally. Agents that inhibit SNCA expression include iRNAagents, ribozymes, and antisense molecules that target SNCA RNA, zincfinger proteins, as well as antibodies or naturally occurring orsynthetic polypeptides, or small molecules, which, in preferredembodiments, bind to and inhibit the SNCA protein.

In a particularly preferred embodiment the inhibitory agent is an iRNAagent that targets an SNCA nucleic acid, e.g., an SNCA RNA. The iRNAagent has an antisense strand complementary to a nucleotide sequence ofan SNCA RNA, and a sense strand sufficiently complementary to hybridizeto the antisense strand. In one embodiment, the iRNA agent includes amodification that stabilizes the iRNA agent in a biological sample. Forexample, the modified iRNA agent is less susceptible to degradation,e.g., less susceptible to cleavage by an exo- or endonuclease. The iRNAagent can include, for example, at least one 5′-uridine-adenine-3′(5′-UA-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide,or at least one 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, whereinthe 5′-uridine is a 2′-modified nucleotide, or at least one5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, or at least one 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide, or at least one 5′-cytidine-cytidine-3′ (5′-CC-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide. TheiRNA agent can include at least 2, at least 3, at least 4 or at least 5of the dinucleotides. In one embodiment, the 2′-modified nucleotide is a2′-O-methylated nucleotide. In another embodiment the iRNA agentincludes a phosphorothioate.

In another embodiment, the antisense strand of the iRNA agent includesthe nucleotide sequence of SEQ ID NOs:6, 16, 18, 20, 22, or 24. Inanother embodiment, the sense strand of the iRNA agent includes thenucleotide sequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23. In yetanother embodiment, the antisense strand of the iRNA agent overlaps thenucleotide sequence of SEQ ID NOs:6, 16, 18, 20, 22, or 24, e.g., by atleast 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 nucleotides. Likewise, the sense strand of the iRNA agent can overlapthe nucleotide sequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23, e.g., byat least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 nucleotides.

In another embodiment, the iRNA agent targets a wildtype SNCA nucleicacid, and in yet another embodiment, the iRNA agent targets apolymorphism or mutation of SNCA. For example, the iRNA agent can targeta mutation in a codon of the SNCA open reading frame that corresponds toan A53T, A30P, or E46K mutation. In some embodiments, the iRNA agenttargets the 3′UTR or the 5′UTR of SNCA. In some embodiment, the iRNAagent targets a spliced isoform of SNCA. For example, the iRNA agent cantarget the splice junction between exons 2 and 4 to downregulateexpression of the 128 amino acid isoform, or the iRNA agent can targetthe splice junction between exons 4 and 6 to target the 112 amino acidisoform.

In some embodiments, the subject (e.g., the human) carries amultiplication (e.g., a duplication or triplication) of the SNCA gene,or a genetic variation in the Parkin or ubiquitin carboxy-terminalhydrolase L1 (UCHL1) gene. In another embodiment, the subject isdiagnosed with a synucleinopathy. The synucleinopathy is characterizedby the aggregation of alpha-synuclein monomers. An iRNA agent can beadministered to a human diagnosed as having, e.g., Parkinson's disease(PD), Alzheimer's disease, multiple system atrophy, Lewy body dementia,or a retinal disorder, e.g., a retinopathy.

In another embodiment, the iRNA agent is at least 21 nucleotides longand includes a sense RNA strand and an antisense RNA strand, wherein theantisense RNA strand is 25 or fewer nucleotides in length, and theduplex region of the iRNA agent is 18-25 nucleotides in length. The iRNAagent may further include a nucleotide overhang having 1 to 4 unpairednucleotides, and the unpaired nucleotides may have at least onephosphorothioate dinucleotide linkage. The nucleotide overhang can be,e.g., at the 3′ end of the antisense strand of the iRNA agent.

In another aspect, the invention features an iRNA agent that targets anSNCA nucleic acid, e.g., an SNCA RNA. The iRNA agent has an antisensestrand complementary to a nucleotide sequence of an SNCA RNA, and asense strand sufficiently complementary to hybridize to the antisensestrand. In one embodiment, the iRNA agent includes a modification thatstabilizes the iRNA agent in a biological sample. For example, themodified iRNA agent is less susceptible to degradation, e.g., lesssusceptible to cleavage by an exo- or endonuclease. In anotherembodiment, the iRNA agent comprises a phosphorothioate or2′-O-methylated (2′-O-Me) nucleotide. The iRNA agent can include, forexample, at least one 5′-uridine-adenine-3′ (5′-UA-3′) dinucleotidewherein the uridine is a 2′-modified nucleotide, or at least one5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide, or at least one 5′-cytidine-adenine-3′(5′-CA-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide, or at least one 5′-uridine-uridine-3′ (5′-UU-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide, or atleast one 5′-cytidine-cytidine-3′ (5′-CC-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide. The iRNA agent can include atleast 2, at least 3, at least 4 or at least 5 of the dinucleotides. Inone embodiment, the 2′-modified nucleotide is a 2′-O-methylatednucleotide.

In another embodiment, the iRNA agent is at least 21 nucleotides longand includes a sense RNA strand and an antisense RNA strand, wherein theantisense RNA strand is 25 or fewer nucleotides in length, and theduplex region of the iRNA agent is 18-25 nucleotides in length. The iRNAagent may further include a nucleotide overhang having 1 to 4 unpairednucleotides, and the unpaired nucleotides may have at least onephosphorothioate dinucleotide linkage. The nucleotide overhang can be,e.g., at the 3′ end of the antisense strand of the iRNA agent.

In another embodiment, the antisense strand of the iRNA agent includesthe nucleotide sequence of SEQ ID NOs:6, 16, 18, 20, 22, or 24 (seeTable 1). In another embodiment, the sense strand of the iRNA agentincludes the nucleotide sequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23(see Table 1). In yet another embodiment, the antisense strand of theiRNA agent overlaps the nucleotide sequence of SEQ ID NOs:6, 16, 18, 20,22, or 24, e.g., by at least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, or 24 nucleotides. Likewise, the sense strand of theiRNA agent can overlap the nucleotide sequence of SEQ ID NOs:5, 15, 17,19, 21, or 23, e.g., by at least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 nucleotides.

In another embodiment, the iRNA agent targets a wildtype SNCA nucleicacid, and in yet another embodiment, the iRNA agent targets apolymorphism or mutation of SNCA. For example, the iRNA agent can targeta mutation in a codon of the SNCA open reading frame that corresponds toan A53T, A30P, or E46K mutation (see FIG. 1B). In some embodiments, theiRNA agent targets the 5′UTR or the 3 ′UTR of SNCA. In some embodiment,the iRNA agent targets a spliced isoform of SNCA. For example, the iRNAagent can target the splice junction between exons 2 and 4 todownregulate expression of the 128 amino acid isoform, or the iRNA agentcan target the splice junction between exons 4 and 6 to target the 112amino acid isoform.

The SNCA gene can be a target for treatment methods of neurodegenerativedisease. In one embodiment, an antisense oligonucleotide, ribozyme, orzinc finger protein can be used to inhibit gene expression, or anantibody or small molecule can be used to target an SNCA polypeptide. Acombination of therapies to downregulate SNCA expression and activitycan also be used.

In another aspect, the invention features a pharmaceutical compositionof an inhibitory agent described herein, e.g., an iRNA agent, ribozyme,or antisense molecule which targets SNCA RNA, an antibody or naturallyoccurring or synthetic polypeptide, or small molecule, which preferablybinds to and inhibits the SNCA protein, and a pharmaceuticallyacceptable carrier.

In a particularly preferred embodiment, the pharmaceutical compositionincludes an iRNA agent targeting the SNCA gene and a pharmaceuticallyacceptable carrier. The iRNA agent has an antisense strand complementaryto a nucleotide sequence of an SNCA RNA, and a sense strand sufficientlycomplementary to hybridize to the antisense strand. In one embodiment,the iRNA agent of the pharmaceutical composition includes a modificationthat stabilizes the iRNA agent in a biological sample. For example, themodified iRNA agent is less susceptible to degradation, e.g., lesssusceptible to cleavage by an exo- or endonuclease. The iRNA agent caninclude, for example, at least one 5′-uridine-adenine-3′ (5′-UA-3′)dinucleotide wherein the uridine is a 2′-modified nucleotide, or atleast one 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide, or at least one5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, or at least one 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide, or at least one 5′-cytidine-cytidine-3′ (5′-CC-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide. TheiRNA agent can include at least 2, at least 3, at least 4 or at least 5of the dinucleotides. In one embodiment, the 2′-modified nucleotide is a2′-O-methylated nucleotide. In another embodiment the iRNA agentincludes a phosphorothioate.

In another embodiment, the iRNA agent of the pharmaceutical compositionis at least 21 nucleotides long and includes a sense RNA strand and anantisense RNA strand, wherein the antisense RNA strand is 25 or fewernucleotides in length, and the duplex region of the iRNA agent is 18-25nucleotides in length. The iRNA agent of the composition may furtherinclude a nucleotide overhang having 1 to 4 unpaired nucleotides, andthe unpaired nucleotides may have at least one phosphorothioatedinucleotide linkage. The nucleotide overhang can be, e.g., at the 3′end of the antisense strand of the iRNA agent.

In another embodiment, the antisense strand of the iRNA agent of thepharmaceutical composition includes the nucleotide sequence of SEQ IDNOs:6, 16, 18, 20, 22, or 24. In another embodiment, the sense strand ofthe iRNA agent of the pharmaceutical composition includes the nucleotidesequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23. In yet anotherembodiment, the antisense strand of the iRNA agent overlaps thenucleotide sequence of SEQ ID NOs:6, 16, 18, 20, 22, or 24, e.g., by atleast 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 nucleotides. Likewise, the sense strand of the iRNA agent can overlapthe nucleotide sequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23, e.g., byat least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 nucleotides.

In another embodiment, the iRNA agent targets a wildtype SNCA nucleicacid, and in another embodiment, the iRNA agent targets a polymorphismor mutation of SNCA. For example, the iRNA agent can target a mutationin a codon of the SNCA open reading frame that corresponds to an A53T,A30P, or E46K mutation. In some embodiments, the iRNA agent targets the3′UTR or the 5′UTR of SNCA. In some embodiments, the iRNA agent targetsa spliced isoform of SNCA. For example, the iRNA agent can target thesplice junction between exons 2 and 4 to downregulate expression of the128 amino acid isoform, or the iRNA agent can target the splice junctionbetween exons 4 and 6 to target the 112 amino acid isoform. In anotheraspect, the invention features a method of reducing the amount of SNCAor SNCA RNA in a cell of a subject (e.g., a mammalian subject, such as ahuman). The method includes contacting cell with an agent which inhibitsthe expression of SNCA. The inhibition can be effected at any level,e.g., at the level of transcription, the level of translation, orpost-translationally. Agents which inhibit SNCA expression include iRNAagents and antisense molecules which target SNCA RNA, as well asantibodies or naturally occurring or synthetic polypeptides, or smallmolecules, which, in preferred embodiments, bind to and inhibit the SNCAprotein.

In a particularly preferred embodiment SNCA RNA is reduced by contactinga cell of the subject with an iRNA agent. In one embodiment, the iRNAagent includes a modification that stabilizes the iRNA agent in abiological sample. For example, the modified iRNA agent is lesssusceptible to degradation, e.g., less susceptible to cleavage by anexo- or endonuclease. The iRNA agent can include, for example, at leastone 5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine isa 2′-modified nucleotide, or at least one 5′-uridine-guanine-3′(5′-UG-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide, or at least one 5′-cytidine-adenine-3′ (5′-CA-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, or atleast one 5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide, or at least one5′-cytidine-cytidine-3′ (5′-CC-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide. The iRNA agent can include at least 2, atleast 3, at least 4 or at least 5 of the dinucleotides. In oneembodiment, the 2′-modified nucleotide is a 2′-O-methylated nucleotide.In another embodiment the iRNA agent includes a phosphorothioate.

In another embodiment, the antisense strand of the iRNA agent of thepharmaceutical composition includes the nucleotide sequence of SEQ IDNOs:6, 16, 18, 20, 22, or 24. In another embodiment, the sense strand ofthe iRNA agent of the pharmaceutical composition includes the nucleotidesequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23. In yet anotherembodiment, the antisense strand of the iRNA agent overlaps thenucleotide sequence of SEQ ID NOs:6, 16, 18, 20, 22, or 24, e.g., by atleast 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 nucleotides. Likewise, the sense strand of the iRNA agent can overlapthe nucleotide sequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23, e.g., byat least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 nucleotides.

In another embodiment, the iRNA agent targets a wildtype SNCA nucleicacid, and in another embodiment, the iRNA agent targets a polymorphismor mutation of SNCA. For example, the iRNA agent can target a mutationin a codon of the SNCA open reading frame that corresponds to an A53T,A30P, or E46K mutation. In some embodiments, the iRNA agent targets the3′UTR or the 5′UTR of SNCA. In some embodiments, the iRNA agent targetsa spliced isoform of SNCA. For example, the iRNA agent can target thesplice junction between exons 2 and 4 to downregulate expression of the128 amino acid isoform, or the iRNA agent can target the splice junctionbetween exons 4 and 6 to target the 112 amino acid isoform.

In another embodiment, the iRNA agent is at least 21 nucleotides longand includes a sense RNA strand and an antisense RNA strand, wherein theantisense RNA strand is 25 or fewer nucleotides in length, and theduplex region of the iRNA agent is 18-25 nucleotides in length. The iRNAagent may further include a nucleotide overhang having 1 to 4 unpairednucleotides, and the unpaired nucleotides may have at least onephosphorothioate dinucleotide linkage. The nucleotide overhang can be,e.g., at the 3′ end of the antisense strand of the iRNA agent.

In another aspect, the invention features a method of making an iRNAagent. The method includes selecting a nucleotide sequence of between 18and 25 nucleotides long from the nucleotide sequence of an SNCA mRNA,and synthesizing the iRNA agent. The sense strand of the iRNA agentincludes the nucleotide sequence selected from SNCA RNA, and theantisense strand is sufficiently complementary to hybridize to the sensestrand. In one embodiment, the method further includes administering theiRNA agent to a subject (e.g., a mammalian subject, such as a humansubject) as described herein.

In another aspect, the invention features a method of evaluating anagent, e.g., an agent of a type described herein, such as a smallmolecule (a small molecule has a molecular weight of preferably lessthan 3,000 Daltons, more preferably less than 2,000 Daltons and yet morepreferably of less than 1000 Daltons), antisense, ribozyme, iRNA agent,or protein, polypeptide, or peptide, e.g., a zinc finger protein, forthe ability to inhibit SNCA expression, e.g., an agent that targets anSNCA or SNCA nucleic acid. The method includes: providing a candidateagent and determining, e.g., by the use of one or more of the testsystems described herein, if said candidate agent modulates, e.g.,inhibits, SNCA expression.

In a preferred embodiment the method includes evaluating the agent in afirst test system; and, if a predetermined level of modulation is seen,evaluating the candidate in a second, preferably different, test system.In a particularly preferred embodiment the second test system includesadministering the candidate agent to an animal and evaluating the effectof the candidate agent on SNCA expression in the animal. In a preferredembodiment two test systems are used and the first is a high-throughputsystem, e.g., in such embodiments the first or initial test is used toscreen at least 100, 1,000, or 10,000 times more compounds than is thesecond, preferably animal, system.

A test system can include: contacting the candidate agent with a targetmolecule, e.g., SNCA, an SNCA nucleic acid, e.g., an RNA or DNA,preferably in vitro, and determining if there is an interaction, e.g.,binding of the candidate agent to the target, or modifying the target,e.g., by making or breaking a covalent bond in the target. Modificationis correlated with the ability to modulate SNCA expression. The testsystem can include contacting the candidate agent with a cell andevaluating modulation of SNCA expression. E.g., this can includecontacting the candidate agent with a cell capable of expressing SNCA orSCNA RNA (from an endogenous gene or from an exogenous construct) andevaluating the level of SNCA or SNCA RNA. In another embodiment the testsystem can include contacting the candidate agent with a cell whichexpresses an RNA or protein from an SNCA control region (e.g., an SNCAcontrol region) linked to a heterologous sequence, e.g., a markerprotein, e.g., a fluorescent protein such as GFP, which construct can beeither chromosomal or episomal, and determining the effect on RNA orprotein levels. The test system can also include contacting thecandidate agent, in vitro, with a tissue sample, e.g., a brain tissuesample, e.g., a slice or section, an optical tissue sample, or othersample which includes neural tissue, and evaluating the level of SNCA orSNCA RNA. The test system can include administering the candidate agent,in vivo, to an animal, and evaluating the level of SNCA or SNCA RNA. Inany of these the effect of the candidate agent on SNCA expression caninclude comparing SNCA gene expression with a predetermined standard,e.g., with control, e.g., an untreated cell, tissue or animal. SNCA geneexpression can be compared, e.g., before and after contacting with thecandidate agent. The method allows determining whether the iRNA agent isuseful for inhibiting SNCA gene expression.

In one embodiment, SNCA gene expression can be evaluated by a method toexamine SNCA RNA levels (e.g., Northern blot analysis, RT-PCR, or RNAseprotection assay) or SNCA protein levels (e.g., Western blot).

In one embodiment, e.g., as a second test, the agent is administered toan animal, e.g., a mammal, such as a mouse, rat, rabbit, human, ornon-human primate, and the animal is monitored for an effect of theagent. For example, a tissue of the animal, e.g., a brain tissue orocular tissue, is examined for an effect of the agent on SNCAexpression. The tissue can be examined for the presence of SNCA RNAand/or protein, for example. In one embodiment, the animal is observedto monitor an improvement or stabilization of a cognitive symptom. Theagent can be administered to the animal by any method, e.g., orally, orby intrathecal or parenchymal injection, such as by stereoscopicinjection into the brain.

In a particularly preferred embodiment, the invention features a methodof evaluating an iRNA agent, e.g., an iRNA agent described herein, thattargets an SNCA nucleic acid. The method includes providing an iRNAagent that targets an SNCA nucleic acid (e.g., an SNCA RNA); contactingthe iRNA agent with a cell containing, and capable of expressing, anSNCA gene; and evaluating the effect of the iRNA agent on SNCAexpression, e.g., by comparing SNCA gene expression with a control,e.g., in the cell. SNCA gene expression can be compared, e.g., beforeand after contacting the iRNA agent with the cell. The method allowsdetermining whether the iRNA agent is useful for inhibiting SNCA geneexpression. For example, the iRNA agent can be determined to be usefulfor inhibiting SNCA gene expression if the iRNA agent reduces expressionby a predetermined amount, e.g., by 10, 25, 50, 75, or 90%, e.g., ascompared with a predetermined reference value, e.g., as compared withthe amount of SNCA RNA or protein prior to contacting the iRNA agentwith the cell. The SNCA gene can be endogenously or exogenouslyexpressed.

The methods and compositions featured in the invention, e.g., themethods and iRNA compositions to treat the neurodegenerative disordersdescribed herein, can be used with any dosage and/or formulationdescribed herein, as well as with any route of administration describedherein.

In addition to their presence in the brain, alpha-synuclein polypeptideshave been found in ocular tissues, including the retina and optic nerve.Accordingly, the compositions and methods described herein are suitablefor treating synucleinopathies of the eye or ocular tissues, includingbut not limited to retinopathies.

Thus, in another aspect, the invention features a method of treating asubject by administering an agent which inhibits the expression of SNCAin the eye or in ocular tissue. In a preferred embodiment, the subjectis a mammal, such as a human, e.g., a subject diagnosed as having, or atrisk for developing a synucleinopathy of the eye, e.g., a retinopathy.The inhibition can be effected at any level, e.g., at the level oftranscription, the level of translation, or post-translationally. Agentswhich inhibit SNCA expression include iRNA agents and antisensemolecules which target SNCA RNA, as well as antibodies or naturallyoccurring or synthetic polypeptides, or small molecules, which, inpreferred embodiments, bind to and inhibit the SNCA protein.

In a particularly preferred embodiment the inhibitory agent is an iRNAagent that targets an SNCA nucleic acid, e.g., an SNCA RNA. The iRNAagent has an antisense strand complementary to a nucleotide sequence ofan SNCA RNA, and a sense strand sufficiently complementary to hybridizeto the antisense strand. In one embodiment, the iRNA agent includes amodification that stabilizes the iRNA agent in a biological sample. Forexample, the modified iRNA agent is less susceptible to degradation,e.g., less susceptible to cleavage by an exo- or endonuclease. The iRNAagent can include, for example, at least one 5′-uridine-adenine-3′(5′-UA-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide,or at least one 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, whereinthe 5′-uridine is a 2′-modified nucleotide, or at least one5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, or at least one 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide, or at least one 5′-cytidine-cytidine-3′ (5′-CC-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide. TheiRNA agent can include at least 2, at least 3, at least 4 or at least 5of the dinucleotides. In one embodiment, the 2′-modified nucleotide is a2′-O-methylated nucleotide. In another embodiment the iRNA agentincludes a phosphorothioate.

In another embodiment, the antisense strand of the iRNA agent includesthe nucleotide sequence of SEQ ID NOs:6, 16, 18, 20, 22, or 24. Inanother embodiment, the sense strand of the iRNA agent includes thenucleotide sequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23. In yetanother embodiment, the antisense strand of the iRNA agent overlaps thenucleotide sequence of SEQ ID NOs:6, 16, 18, 20, 22, or 24, e.g., by atleast 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 nucleotides. Likewise, the sense strand of the iRNA agent can overlapthe nucleotide sequence of SEQ ID NOs:5, 15, 17, 19, 21, or 23, e.g., byat least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 nucleotides.

In another embodiment, the iRNA agent targets a wildtype SNCA nucleicacid, and in yet another embodiment, the iRNA agent targets apolymorphism or mutation of SNCA. For example, the iRNA agent can targeta mutation in a codon of the SNCA open reading frame that corresponds toan A53T, A30P, or E46K mutation. In some embodiments, the iRNA agenttargets the 3 ′UTR or the 5′UTR of SNCA. In some embodiment, the iRNAagent targets a spliced isoform of SNCA. For example, the iRNA agent cantarget the splice junction between exons 2 and 4 to downregulateexpression of the 128 amino acid isoform, or the iRNA agent can targetthe splice junction between exons 4 and 6 to target the 112 amino acidisoform.

In some embodiments, the subject (e.g., the human) carries amultiplication (e.g., a duplication or triplication) of the SNCA gene,or a genetic variation in the Parkin or ubiquitin carboxy-terminalhydrolase L1 (UCHL1) gene. In another embodiment, the subject isdiagnosed with a synucleinopathy. The synucleinopathy is characterizedby the aggregation of alpha-synuclein monomers. An iRNA agent can beadministered to a human diagnosed as having, e.g., Parkinson's disease(PD), Alzheimer's disease, multiple system atrophy, Lewy body dementia,or a retinal disorder, e.g., a retinopathy.

In another embodiment, the iRNA agent is at least 21 nucleotides longand includes a sense RNA strand and an antisense RNA strand, wherein theantisense RNA strand is 25 or fewer nucleotides in length, and theduplex region of the iRNA agent is 18-25 nucleotides in length. The iRNAagent may further include a nucleotide overhang having 1 to 4 unpairednucleotides, and the unpaired nucleotides may have at least onephosphorothioate dinucleotide linkage. The nucleotide overhang can be,e.g., at the 3′ end of the antisense strand of the iRNA agent.

A “substantially identical” sequence includes a region of sufficienthomology to the target gene, and is of sufficient length in terms ofnucleotides, that the iRNA agent, or a fragment thereof, can mediatedown regulation of the target gene. Thus, the iRNA agent is or includesa region which is at least partially, and in some embodiments fully,complementary to a target RNA transcript, e.g, the SNCA transcript. Itis not necessary that there be perfect complementarity between the iRNAagent and the target, but the correspondence must be sufficient toenable the iRNA agent, or a cleavage product thereof, to direct sequencespecific silencing, e.g., by RNAi cleavage of the target RNA, e.g.,mRNA. Complementarity, or degree of homology with the target strand, ismost critical in the antisense strand. While perfect complementarity,particularly in the antisense strand, is often desired some embodimentscan include, particularly in the antisense strand, one or more butpreferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to thetarget RNA). The mismatches, particularly in the antisense strand, aremost tolerated in the terminal regions and if present are preferably ina terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5′ and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the overall doublestrand character of the molecule.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogates, all of which are described herein or are wellknown in the RNA synthetic art. While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those that have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a5′-modification which includes one or more phosphate groups or one ormore analogs of a phosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can downregulate the expression of atarget gene, e.g., an SNCA gene. While not wishing to be bound bytheory, an iRNA agent may act by one or more of a number of mechanisms,including post-transcriptional cleavage of a target mRNA sometimesreferred to in the art as RNAi, or pre-transcriptional orpre-translational mechanisms. An iRNA agent can include a single strandor can include more than one strands, e.g., it can be a double stranded(ds) iRNA agent. If the iRNA agent is a single strand it is particularlypreferred that it include a 5′ modification which includes one or morephosphate groups or one or more analogs of a phosphate group.

An iRNA agent that targets an SNCA nucleic acid can be referred to as ananti-SNCA iRNA agent.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thisdescription, and from the claims. This application incorporates allcited references, patents, and patent applications by references intheir entirety for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is the sequence of the full length mRNA of human SNCA(transcript variant NACP140; GenBank Access. No. NM_(—)000345; SEQ IDNO:1). The start and stop codons of the open reading frame are denotedin bold and italics. Sequences targeted by the siRNAs SNCA1, 3, 4, 5, 6,and 9 are underlined. Sequences of the siRNAs SNCA2, 7 and 8 are shadedin gray. SNCA1 targets nucleotides 197-217; SNCA2 targets nucleotides205-225; SNCA3 targets nucleotides 308-330; SNCA4 targets nucleotides231-251; SNCA5 targets nucleotides 356-376; SNCA6 targets nucleotides261-279; SNCA7 targets nucleotides 403-421; SNCA8 targets nucleotides451-469; SNCA9 targets nucleotides 1311-1329. Brackets flank the twoalternative internal exons (exons 3 and 5).

FIG. 1B is the sequence of the full length protein of human SNCA(transcript variant NACP140; GenBankAccess. No. NM_(—)000345; SEQ IDNO:2).

FIG. 2 is a Western blot of EGFP or EGFP/NACP fusion proteins expressedin BE(2)-M17 human neuroblastoma cells. The cells were cotransfectedwith a vector expressing EGFP or EGFP/NACP fusion protein and an siRNAlisted in Table 1. In the figure, siRNAs Mayo1, Mayo2, Mayo3, Mayo4,Mayo5, Mayo6, Mayo7, Mayo8, and Mayo9, are equivalent to the siRNAs ofTable 1 (SNCA1, SNCA2, SNCA3, SNCA4, SNCA5, SNCA6, SNCA7, SNCA8, andSNCA9, respectively). Control experiments included cotransfection of theEGFP and EGFP/NACP vectors with the siRNA Mr control dsRNA (vector andα-syn lanes labeled “siRNA Mr”), transfection of EGFP and EGFP/NACPvectors without dsRNA, and untransfected cells. The sequence of siRNA Mris provided in Table 1.

FIG. 3 is a Western blot detecting EGFP/NACP fusion proteins expressedin BE(2)-M17 human neuroblastoma cells. The cells were cotransfectedwith a vector expressing an EGFP/NACP fusion protein and varying nMconcentrations of Mayo2, Mayo7, or Mayo8 siRNA. These siRNAs areequivalent to siRNAs SNCA2, SNCA7, and SNCA8, of Table 1 respectively.The Western blots were stripped of the anti-GFP antibody, and reprobedwith anti-tubulin antibody to monitor equivalent loading of proteinbetween samples.

FIG. 4 is a Western blot detecting EGFP/NACP fusion expressed inBE(2)-M17 human neuroblastoma cells. The cells were cotransfected with avector expressing an EGFP/NACP fusion protein and 50 nM Mayo2, Mayo7, orMayo8 siRNA. These siRNAs are equivalent to siRNAs SNCA2, SNCA7, andSNCA8 of Table 1, respectively. In the “control” experiment, no siRNAwas transfected with the fusion construct. Protein expression wasmonitored over the course of six days. The Western blots were strippedof the anti-GFP antibody, and reprobed with anti-tubulin antibody tomonitor equivalent loading of protein between samples. The cells werenon-dividing.

FIG. 5 is a graph depicting relative alpha-synuclein levels assayed byWestern blot analysis. Neuroblastoma cells were transfected with Mayo2,Mayo7, or Mayo8 siRNA. Endogenous alpha synuclein protein expression wasmonitored over the course of three days. In the “untransfected”experiment, no siRNA was transfected, and protein levels in thesesamples were set as 100% normal expression.

FIG. 6 is a graph depicting the effect of Mayo2, 7, and 8 siRNAs onlevels of endogenous alpha-synuclein RNA.

FIG. 7 is a graph depicting the activity of the siRNAs on human andmouse alpha-synuclein/EGFP conjugate expression. BE(2)-M17 humanneuroblastoma cells were cotransfected with a plasmid encoding EGFP(vector) or EGFP conjugated to either human or mouse alpha-synuclein andMayo2, Mayo7 or Mayo8. Expression was equalized using tubulinimmunoreactivity and measured as a proportion of the expression of theplasmid-only EGFP immunoreactivity (control).

FIG. 8A is a polyacrylamide gel depicting a T1 mapping experiment ofcleavage sites in Mayo7 (also called AL-DUP-1477) and Mayo8 (also calledAL-DUP-1478) siRNAs. Lanes 1 are controls and represent siRNA incubatedin T1 buffer; lanes 2 represent siRNA incubated in 1×T1 RNAse; lanes 3represent siRNA incubated in 0.1×T1 RNAse; lanes 4 are an alkalineladder; lanes 5 are Mayo7 and Mayo8, respectively, incubated in humanserum for four hours; lanes 6 are Mayo7 and Mayo8, respectively, priorto incubation in human serum. *s indicates that the siRNA was 5′³²P-labeled on the sense strand by incubating with T4 Polynucleotidekinase and gamma-³²P-ATP.

FIG. 8B is an illustration of the sites of siRNA cleavage following theT1 RNAse assay. The cleavage sites include sites of T1 cleavage (3′ ofG) and cleavage from nucleases in the human serum.

FIG. 9A is a Western blot of EGFP or EGFP/NACP fusion proteins expressedin a neuroblastoma cell line. The cells were cotransfected with either aplasmid expressing EGFP, or a plasmid expressing EGFP/NACP fusionprotein, and an siRNA listed in Table 1. In the figure, siRNAs Mayo2,Mayo7, Mayo7s, Mayo8, Mayo8s1, and Mayo8s2 are equivalent to the siRNAsof Table 1 (SNCA2, SNCA7, SNCA7s, SNCA8, SNCA8s1, and SNCA8s2,respectively). In the control experiment, siRNA was not transfected intocells with the EGFP and EGFP-NACP vectors. The Western blots werestripped of the anti-GFP antibody, and reprobed with anti-tubulinantibody to monitor equivalent loading of protein between samples.

FIG. 9B is a graph depicting the effect of the dsRNAs of FIG. 9A onEGFP/alpha-synuclein protein expression. The y-axis represents percentEGFP immunoreactivity (IR) compared to the control untransfectedexperiment (untrn).

FIG. 10A is a polyacrylamide gel demonstrating the stability of SNCA8siRNA (see Table 1). The RNA in the gel is detected with Stains-All(Sigma, St. Louis, Mo.). Lane 1 is SNCA8 siRNA duplex. Lane 2 is SNCA8siRNA in PBS control at 0 hour time point. Lane 3 is SNCA8 siRNA in PBScontrol at 24 hour time point. Lane 4 is SNCA8 siRNA in human serum at 0hour time point. Lane 5 is SNCA8 siRNA in human serum followingincubation for 30 minutes. Lane 6 is SNCA8 siRNA in human serumfollowing incubation for 4 hours. Lane 7 is SNCA8 siRNA in human serumfollowing incubation for 24 hours.

FIG. 10B is a polyacrylamide gel demonstrating the stability of SNCA8siRNA (see Table 1). The RNA in the gel is detected with Stains-All(Sigma, St. Louis, Mo.). Lane 1 is SNCA8s1 siRNA duplex. Lane 2 isSNCA8s1 siRNA in PBS control at 0 hour time point. Lane 3 is SNCA8s1siRNA in PBS control at 24 hour time point. Lane 4 is SNCA8s1 siRNA inhuman serum at 0 hour time point. Lane 5 is SNCA8s1 siRNA in human serumfollowing incubation for 30 minutes. Lane 6 is SNCA8s1 siRNA in humanserum following incubation for 4 hours. Lane 7 is SNCA8s1 siRNA in humanserum following incubation for 24 hours.

FIG. 10C is a polyacrylamide gel demonstrating the stability of SNCA8siRNA (see Table 1). The RNA in the gel is detected with Stains-All(Sigma, St. Louis, Mo.). Lane 1 is SNCA8s2 siRNA duplex. Lane 2 isSNCA8s2 siRNA in PBS control at 0 hour time point. Lane 3 is SNCA8s2siRNA in PBS control at 24 hour time point. Lane 4 is SNCA8s2 siRNA inhuman serum at 0 hour time point. Lane 5 is SNCA8s2 siRNA in human serumfollowing incubation for 30 minutes. Lane 6 is SNCA8s2 siRNA in humanserum following incubation for 4 hours. Lane 7 is SNCA8s2 siRNA in humanserum following incubation for 24 hours.

FIG. 11 is a graph demonstrating the gene specificity of Mayo2.

FIG. 12A is a graph demonstrating the effect of siRNA on SNCA RNAexpression in mouse brain tissue. E1, E2, and E3 represent results fromthree different mice injected with 2 uL of 200 uM Mayo-8s2m siRNA. C1,C2, and C3 represent results from three different mice injected with 2uL phosphate buffered saline. Avg-E is the average α-synuclein/18S rRNAratio of E1, E2, and E3. Avg-C is the average α-synuclein/18S rRNA ratioof the three control samples. The 18S rRNA control was amplified in anRT-PCR reaction performed in a separate tube from the α-synuclein RT-PCRreaction.

FIG. 12B is a graph demonstrating the effect of siRNA on SNCA RNAexpression in the same mouse brain tissue described in FIG. 2A. In theseRT-PCR reactions, the 18S rRNA control was amplified in an RT-PCRreaction performed in the same tube as the α-synuclein RT-PCR reaction.

FIG. 13 is a graph showing silencing of endogenous alpha-synuclein byintraparenchymal infusion of siRNA.

FIG. 14 shows synuclein expression in cortex of siRNA treated mice inthe injected and non-injected sides of the brain.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNAthrough a process known as RNA interference (RNAi). The process occursin a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA aresequence-specific mediators of RNA silencing, e.g., by causing RNAdegradation. While not wishing to be bound by theory, it may be that amolecular signal, which may be merely the specific length of thefragments, present in these 21-23 nt fragments, recruits cellularfactors that mediate RNAi. Described herein are methods for preparingand administering these 21-23 nt fragments, and other iRNA agents, andtheir use for specifically inactivating gene function, and the functionof the SNCA gene in particular. The use of iRNA agents (or recombinantlyproduced or chemically synthesized oligonucleotides of the same orsimilar nature) enables the targeting of specific mRNAs for silencing inmammalian cells. In addition, longer dsRNA agent fragments can also beused, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferonresponse which is frequently deleterious, short dsRNAs (sRNAs) do nottrigger the interferon response, at least not to an extent that isdeleterious to the cell and host. In particular, the length of the iRNAagent strands in an sRNA agent can be less than 31, 30, 28, 25, or 23nt, e.g., sufficiently short to avoid inducing a deleterious interferonresponse. Thus, the administration of a composition of sRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of a target gene while circumventing the interferonresponse. Further, use of a discrete species of iRNA agent can be usedto selectively target one allele of a target gene, e.g., in a subjectheterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNAagent that disrupts a component of the interferon response, e.g.,dsRNA-activated protein kinase PKR. Such a cell can be treated with asecond iRNA agent that includes a sequence complementary to a target RNAand that has a length that might otherwise trigger the interferonresponse.

As used herein, a “subject” refers to a mammalian organism undergoingtreatment for a disorder mediated by SNCA expression. The subject can bea mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate.In a preferred embodiment, the subject is a human.

As used herein, disorders associated with SNCA expression refers to anybiological or pathological state that (1) is mediated in part by thepresence of SNCA protein and (2) whose outcome can be affected byreducing the level of SNCA protein present. Specific disordersassociated with SNCA expression are noted below.

Because iRNA agent mediated silencing can persist for several days afteradministering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen.

Alpha-synuclein

Alpha-synuclein protein is primarily found in the cytoplasm, but hasalso been localized to the nucleus. In dopaminergic neurons,alpha-synuclein is membrane bound. The protein is a soluble monomernormally localized at the presynaptic region of axons. The protein canform filamentous aggregates that are the major component ofintracellular inclusions in neurodegenerative synucleinopathies.

Alpha-synuclein protein is associated with a number of diseasescharacterized by synucleinopathies. Three point mutations (A53T, A30Pand E46K), and SNCA duplication and triplication events are linked toautosomal dominant Parkinson's disease (familial PD, also called FPD).The A53T and A30P mutations cause configuration changes in the SNCAprotein that promote in vitro protofibril formation. The triplicationevent results in a two-fold overexpression of SNCA protein.Alpha-synuclein is a major fibrillar component of Lewy bodies, thecytoplasmic inclusions that are characteristic of FPD and idiopathic PD,and the substantia nigra of a Parkinson's disease brain is characterizedby fibrillar alpha-synuclein. In Alzheimer's patients, SNCA peptides area major component of amyloid plaques in the brains of patients withAlzheimer's disease.

Aggregation of alpha-synuclein in the cytoplasm of cells can be causedby a number of mechanisms, including overexpression of the protein,inhibition of protein degradation, or a mutation that affects thestructure of the protein, resulting in an increased tendency of theprotein to self-associate.

An SNCA gene product can be a target for treatment methods ofneurodegenerative diseases such as PD. The treatment methods can includetargeting of an SNCA nucleic acid with an iRNA agent. Alternatively, oradditionally, an antisense RNA can be used to inhibit gene expression,or an antibody or small molecule can be used to target an SNCA nucleicacid. In general, an antisense RNA, anti-SNCA antibody, or smallmolecule can be used in place of an iRNA agent, e.g., by any of themethods or compositions described herein. A combination of therapies todownregulate SNCA expression and activity can also be used.

Sequencing of the SNCA gene has revealed common variants including adinucleotide repeat sequence (REP1) within the promoter. REP1 varies inlength across populations, and certain allelic variants are associatedwith an increased risk for PD (Krüger et al., Ann Neurol. 45:611-7,1999). The SNCA gene REP1 locus is necessary for normal gene expression(Touchman et al., Genome Res. 11:78-86, 2001). SNCA gene expressionlevels among the different REP1 alleles varied significantly over a3-fold range, suggesting that the association of specific genotypes withan increased risk for PD may be a consequence of SNCA geneover-expression (Chiba-Falek and Nussbaum, Hum Mol Genet. 10:3101-9,2001). Functional analysis of intra-allelic variation at the SNCA geneREP1 locus implied that overall length of the allele plays the main rolein transcriptional regulation; sequence heterogeneity is unlikely toconfound genetic association studies based on alleles defined by length(Chiba-Falek et al., Hum Genet. 113:426-31, 2003). The recent discoveryof SNCA gene triplication as a rare cause of PD is consistent with theobservation that polymorphism within the gene promoter conferssusceptibility via the same mechanism of gene over-expression (Singletonet al., Science 302:841, 2003).

Three splice variants of SNCA have been identified (see FIG. 1A). Thefull-length 140 amino acid protein is the most abundant form. A 128amino acid form lacks exon 3, and a 112 amino acid form lacks exon 5. AniRNA of the invention can target any isoform of SNCA. An iRNA can targeta common exon (e.g., exon 2, 4, 6, or 7) to effectively target all knownisoforms. An iRNA agent can target a splice junction or an alternativelyspliced exon to target specific isoforms. For example, to target the 112amino acid isoform, an iRNA agent can target an mRNA sequence thatoverlaps the exon 4/exon 6 splice junction. To target the 128 amino acidprotein isoform, an iRNA agent can target an mRNA sequence that overlapsthe exon 2/exon 4 junction.

Treatment of Parkinson's Disease

Any patient having PD (or any other alpha-synuclein related disorder),is a candidate for treatment with a method or composition describedherein. Preferably the patient is not terminally ill (e.g., the patienthas life expectancy of two years or more), and preferably the patienthas not reached end-stage Parkinson's disease (i.e., Hoehn and Yahrstage 5).

Presymptomatic subjects can also be candidates for treatment with ananti-SNCA agent, e.g., an anti-SNCA iRNA agent, antisenseoligonucleotide, ribozyme, zinc finger protein, antibody, or smallmolecule. In one embodiment, a presymptomatic candidate is identified byeither or both of risk-factor profiling and functional neuroimaging(e.g., by fluorodopa and positron emission tomography). For example, thecandidate can be identified by risk-factor profiling followed byfunctional neuroimaging.

Individuals having any genotype are candidates for treatment. In someembodiments the patient will carry a particular genetic mutation thatplaces the patient at increased risk for developing PD. For example, anindividual carrying an SNCA gene multiplication, e.g., an SNCA geneduplication or triplication is at increased risk for developing PD andis a candidate for treatment with the iRNA agent. In addition, again-of-function mutation in SNCA can increase an individual's risk fordeveloping PD. An individual carrying an SNCA REP1 genotype (e.g., aREP1 “+1 allele” heterozygous or homozygous genotype) can be a candidatefor such treatment. An individual homozygous for the REP1 +1 alleleoverexpresses SNCA. An individual carrying a mutation in the UCHL-1,parkin, or SNCA gene is at increased risk for PD and can be a candidatefor treatment with an anti-SNCA iRNA agent. Particularly, a mutation inthe UCHL-1 or parkin gene will cause a decrease in gene or proteinactivity. An individual carrying a Tau genotype (e.g., a mutation in theTau gene) or a Tau haplotype, such as the H1 haplotype is also at riskfor developing PD. Other genetic risk factors include mutations in theMAPT, DJ1, PINK1, and NURR1 genes, and polymorphism in several genesincluding the SNCA, parkin, MAPT, and NAT2 genes.

Non-genetic (e.g., environmental) risk factors for PD include age (e.g.,over age 30, 35, 40, 45, or 50 years), gender (men are generally have ahigher risk than women), pesticide exposure, heavy metal exposure, andhead trauma. In general, exogenous and endogenous factors that disruptthe ubiquitin proteasomal pathway or more specifically inhibit theproteasome, or which disrupt mitochondrial function, or which yieldoxidative stress, or which promote the aggregation and fibrillization ofalpha-synuclein, can increase the risk of an individual for developingPD, and can contribute to the pathogenesis of PD.

In one embodiment, an iRNA agent can be used to target wildtype SNCA insubjects with PD.

Treatment of Other Neurodegenerative Disorders

Any disease characterized by a synucleinopathy can be treated with aninhibitory agent described herein (e.g., an agent that targets SNCA),including Lewy body dementia, Multiple System Atrophy, and Alzheimer'sDisease. Individuals having any genotype are candidates for treatment.In some embodiments, the patient will carry a particular geneticmutation that places them at increased risk for developing asynucleinopathy.

In one embodiment, an iRNA agent can be used to target wildtype SNCA insubjects with a neurodegenerative disorder.

An individual can develop a synucleinopathy as a result of certainenvironmental factors. For example, oxidative stress, certain pesticides(e.g., 24D and agent orange), bacterial infection, and head trauma havebeen linked to an increase in the risk of developing PD, and can bedetermining factors for determining the risk of an individual forsynucleinopathies. These factors (and others disclosed herein) can beconsidered when evaluating the risk profile of a candidate subject foranti-SNCA therapy.

Design and Selection of iRNA Agents

Candidate iRNA agents can be designed by performing, for example, a genewalk analysis. Overlapping, adjacent, or closely spaced candidate agentscorresponding to all or some of the transcribed region can be generatedand tested. Each of the iRNA agents can be tested and evaluated for theability to down regulate target gene expression (see below, “Evaluationof Candidate iRNA agents”).

An iRNA agent can be rationally designed based on sequence informationand desired characteristics. For example, an iRNA agent can be designedaccording to the relative melting temperature of the candidate duplex.Generally, the duplex will have a lower melting temperature at the 5′end of the antisense strand than at the 3′ end of the antisense strand.This and other elements of rational design are discussed in greaterdetail below (see, e.g., sections labeled “”Palindromes,” “Asymmetry,”and “Z-X-Y,” and “Differential Modification of Terminal DuplexStability” and “Other-than-Watson-Crick Pairing.”

An iRNA agent targeting an SNCA RNA can have the sequences of any of thesiRNAs of Table 1. In particular, the iRNA agent can have the sequenceof SNCA2, 7 (or 7s), or 8 (or 8s1 or 8s2), which were found to be themost effective for silencing the SNCA gene in vivo and in vitro.

Evaluation of Candidate iRNA Agents

A candidate anti-SNCA iRNA agent can be evaluated for its ability todown-regulate SNCA gene expression. For example, a candidate iRNA agentcan be provided, and contacting with a cell that expresses the SNCAgene. The level of SNCA gene expression prior to and following contactwith the candidate iRNA agent can be compared. The SNCA target gene canbe an endogenous or exogenous gene within the cell. If it is determinedthat the amount of RNA or protein expressed from the SNCA gene is lowerfollowing contact with the iRNA agent, then it can be concluded that theiRNA agent downregulates SNCA gene expression. The level of SNCA RNA orprotein in the cell can be determined by any method desired. Forexample, the level of SNCA RNA can be determined by Northern blotanalysis, reverse transcription coupled with polymerase chain reaction(RT-PCR), or RNAse protection assay. The level of protein an bedetermined by, for example, Western blot analysis.

The iRNA agent can be tested in an in vitro or/and in an in vivo system.For example, the target gene or a fragment thereof can be fused to areporter gene on a plasmid. The plasmid can be transfected into a cellwith a candidate iRNA agent. The efficacy of the iRNA agent can beevaluated by monitoring expression of the reporter gene. The reportergene can be monitored in vivo, such as by fluorescence or in situhybridization. Exemplary fluorescent reporter genes include but are notlimited to green fluorescent protein and luciferase. Expression of thereporter gene can also be monitored by Northern blot, RT-PCR,RNAse-protection assay, or Western blot analysis as described above.

Efficacy of an anti-SNCA iRNA agent can be tested in a mammalian cellline (e.g., a mammalian neural cell line), such as a human neuroblastomacell line. For example, a cell line useful for testing efficacy of ananti-SNCA iRNA agent are those with a neuronal phenotype(neuroblastomas, neuronally differentiated phaeochromocytomas andprimary neuronal cultures) or non neuronal cell lines (e.g. kidney,muscle or ovarian cells). Neuroblastoma cell lines include BE(2)-M17,SH-SY5Y (both human) and N2a (mouse). BE(2)-M17 cells biochemicallymimic dopaminergic neurons of the human brain affected byalpha-synucleinopathies.

Controls include

-   -   (1) testing the efficacy and specificity of an iRNA by assaying        for a decrease in expression of the target gene by, for example,        comparison to expression of an endogenous or exogenous        off-target RNA or protein; and    -   (2) testing specificity of the effect on target gene expression        by administering a “nonfunctional” iRNA agent.

Nonfunctional control iRNA agents can

-   -   (a) target a gene not expressed in the cell;    -   (b) be of nonsensical sequence (e.g., a scrambled version of the        test iRNA); or    -   (c) have a sequence complementary to the target gene, but be        known by previous experiments to lack an ability to silence gene        expression.

Assays include time course experiments to monitor stability and durationof silencing effect by an iRNA agent and monitoring in dividing versusnondividing cells. Presumably in dividing cells, the dsRNA is dilutedout over time, thus decreasing the duration of the silencing effect. Theimplication is that dosage will have to be adjusted in vivo, and/or aniRNA agent will have to be administered more frequently to maintain thesilencing effect. To monitor nondividing cells, cells can be arrested byserum withdrawal. Neurons are post-mitotic cells, and thus neural cellsare aptly suited for assaying the stability of iRNA agents, such as ananti-SNCA iRNA agent, for use in therapeutic compositions for thetreatment of disorders of the nervous system, e.g., neurodegenerativedisorders.

A candidate iRNA agent can also be evaluated for cross-speciesreactivity. For example, cell lines derived from different species(e.g., mouse vs. human) or in biological samples (e.g., serum or tissueextracts) isolated from different species can be transfected with atarget iRNA agent and a candidate iRNA agent. The efficacy of the iRNAagent can be determined for the cell from the different species.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to itssusceptibility to cleavage by an endonuclease or exonuclease, such aswhen the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject. The component (e.g., an exonuclease or endonuclease) canbe specific for a particular area of the body, such as a particulartissue, organ, or bodily fluid (e.g., blood, plasma, or serum). Sites inan iRNA agent that are susceptible to cleavage, either byendonucleolytic or exonucleolytic cleavage, in certain areas of thebody, may be resistant to cleavage in other areas of the body. Anexemplary method includes:

(1) determining the point or points at which a substance present in thebody of a subject, and preferably a component present in a compartmentof the body into which a therapeutic dsRNA is to be introduced (thisincludes compartments into which the therapeutic is directly introduced,e.g., the circulation, as well as in compartments to which thetherapeutic is eventually targeted; in some cases, e.g, the eye or thebrain the two are the same), cleaves a dsRNA, e.g., an iRNA agent, and

(2) identifying one or more points of cleavage, e.g., endonucleolytic,exonucleolytic, or both, in the dsRNA. Optionally, the method furtherincludes providing an RNA modified to inhibit cleavage at such sites.

These steps can be accomplished by using one or more of the followingassays:

-   -   (i) (a) contacting a candidate dsRNA, e.g., an iRNA agent, with        a test agent (e.g., a biological agent),        -   (b) using a size-based assay, e.g., gel electrophoresis to            determine if the iRNA agent is cleaved. In a preferred            embodiment a time course is taken and a number of samples            incubated for different times are applied to the size-based            assay. In preferred embodiments, the candidate dsRNA is not            labeled. The method can be a “stains all” method.    -   (ii) (a) supplying a candidate dsRNA, e.g., an iRNA agent, which        is radiolabeled;        -   (b) contacting the candidate dsRNA with a test agent,        -   (c) using a size-based assay, e.g., gel electrophoresis to            determine if the iRNA agent is cleaved. In a preferred            embodiment a time course is taken where a number of samples            are incubated for different times and applied to the            size-based assay. In preferred embodiments, the            determination is made under conditions that allow            determination of the number of nucleotides present in a            fragment. E.g., an incubated sample is run on a gel having            markers that allow assignment of the length of cleavage            products. The gel can include a standard that is a “ladder”            digestion. Either the sense or antisense strand can be            labeled. Preferably only one strand is labeled in a            particular experiment. The label can be incorporated at the            5′ end, 3′ end, or at an internal position. Length of a            fragment (and thus the point of cleavage) can be determined            from the size of the fragment based on the ladder and            mapping using a site-specific endonuclease such as RNAse T1.    -   (iii) fragments produced by any method, e.g., one of those        above, can be analyzed by mass spectrometry. Following        contacting the iRNA with the test agent, the iRNA can be        purified (e.g., partially purified), such as by        phenol-chloroform extraction followed by precipitation. Liquid        chromatography can then be used to separate the fragments and        mass spectrometry can be used to determine the mass of each        fragment. This allows determination of the mechanism of        cleavage, e.g., if by direct phosphate cleavage, such as be 5′        or 3′ exonuclease cleavage, or mediated by the 2′OH via        formation of a cyclic phosphate.

More than one dsRNA, e.g., anti-SNCA iRNA agent, can be evaluated. Theevaluation can be used to select a sequence for use in a therapeuticiRNA agent. For example, it allows the selection of a sequence having anoptimal (usually minimized) number of sites that are cleaved by asubstance(s), e.g., an enzyme, present in the relevant compartments of asubject's body. Two or more dsRNA candidates can be evaluated to selecta sequence that is optimized. For example, two or more candidates can beevaluated and the one with optimum properties, e.g., fewer cleavagesites, selected.

The information relating to a site of cleavage can be used to select abackbone atom, a sugar or a base, for modification, e.g., a modificationto decrease cleavage.

Exemplary modifications include modifications that inhibitendonucleolytic degradation, including the modifications describedherein. Particularly favored modifications include: 2′ modification,e.g., provision of a 2′ OMe moiety on a U in a sense or antisensestrand, but especially on a sense strand; modification of the backbone,e.g., with the replacement of an O with an S, in the phosphate backbone,e.g., the provision of a phosphorothioate modification, on the U or theA or both, especially on an antisense strand; replacement of the U witha C5 amino linker; replacement of an A with a G (sequence changes arepreferred to be located on the sense strand and not the antisensestrand); and modification of the at the 2′, 6′, 7′, or 8′ position.Preferred embodiments are those in which one or more of thesemodifications are present on the sense but not the antisense strand, orembodiments where the antisense strand has fewer of such modifications.

Exemplary modifications also include those that inhibit degradation byexonucleases. Examples of modifications that inhibit exonucleolyticdegradation can be found herein. Particularly favored modificationsinclude: 2′ modification, e.g., provision of a 2′ OMe moiety in a 3′overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom ofthe molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′position, as indicated by the context); modification of the backbone,e.g., with the replacement of a P with an S, e.g., the provision of aphosphorothioate modification, or the use of a methylated P in a 3′overhang, e.g., at the 3′ terminus; combination of a 2′ modification,e.g., provision of a 2′ O Me moiety and modification of the backbone,e.g., with the replacement of a P with an S, e.g., the provision of aphosphorothioate modification, or the use of a methylated P, in a 3′overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl;modification with an abasic pyrrolidine in a 3′ overhang, e.g., at the3′ terminus; modification with naproxen, ibuprofen, or other moietieswhich inhibit degradation at the 3′ terminus.

These methods can be used to select and or optimize a therapeuticanti-SNCA iRNA agent.

The method can be used to evaluate a candidate dsRNA, e.g., a candidateiRNA agent, which is unmodified or which includes a modification, e.g.,a modification that inhibits degradation, targets the dsRNA molecule, ormodulates hybridization. Such modifications are described herein. Acleavage assay can be combined with an assay to determine the ability ofa modified or non-modified candidate to silence the target. E.g., onemight (optionally) test a candidate to evaluate its ability to silence atarget (or off-target sequence), evaluate its susceptibility tocleavage, modify it (e.g., as described herein, e.g., to inhibitdegradation) to produce a modified candidate, and test the modifiedcandidate for one or both of the ability to silence and the ability toresist degradation. The procedure can be repeated. Modifications can beintroduced one at a time or in groups. A cell-based method can be usedto monitor the ability of the iRNA agent to silence. This can befollowed by a different method, e.g, a whole animal method, to confirmactivity.

A test agent refers to a biological agent, e.g., biological sample,tissue extract or prep, serum, a known enzyme or other molecule known tomodify, e.g., cleave, a dsRNA, e.g., an endonuclease. The test agent canbe in a compartment of the body in which the RNAi agent will be exposed.For example; for an iRNA agent that is administered directly in toneural tissue (e.g., into the brain or into the spinal cord) the testagent could be brain tissue extract or spinal fluid. An iRNA agent thatis to be supplied directly to the eye can be incubated with an extractof the eye.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting SNCA geneexpression can be tested for functionality in vivo in an animal model(e.g., in a mammal, such as in mouse or rat). For example, the iRNAagent can be administered to an animal, and the iRNA agent evaluatedwith respect to its biodistribution, stability, and its ability toinhibit SNCA gene expression.

The iRNA agent can be administered directly to the target tissue, suchas by injection, or the iRNA agent can be administered to the animalmodel in the same manner that it would be administered to a human. Forexample, the iRNA agent can be injected directly into a target region ofthe brain (e.g., into the cortex, the substantia nigra, the globuspallidus, or the hippocampus), and after a period of time, the brain canbe harvested and tissue slices examined for distribution of the agent.

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker such as fluorescein; aradioactive label, such as ³²P, ³³P, or ³H; gold particles; or antigenparticles for immunohistochemistry).

An iRNA agent useful for monitoring biodistribution can lack genesilencing activity in vivo. For example, the iRNA agent can target agene not present in the animal (e.g., an iRNA agent injected into mousecan target luciferase), or an iRNA agent can have a non-sense sequence,which does not target any gene, e.g., any endogenous gene).Localization/biodistribution of the iRNA can be monitored by a traceablelabel attached to the iRNA agent, such as a traceable agent describedabove

The iRNA agent can be evaluated with respect to its ability to downregulate SNCA expression. Levels of SNCA expression in vivo can bemeasured, for example, by in situ hybridization, or by the isolation ofRNA from tissue prior to and following exposure to the iRNA agent. SNCARNA can be detected by any desired method, including but not limited toRT-PCR, Northern blot, or RNAase protection assay. Alternatively, oradditionally, SNCA gene expression can be monitored by performingWestern blot analysis on tissue extracts treated with the anti-SNCA iRNAagent.

An anti-SNCA iRNA agent can be tested in a mouse model for PD, such as amouse carrying a wildtype copy of the human SNCA gene (Masliah et al.,Science 287: 1265-1269, 2000) or in mouse carrying a mutant human SNCA(Richfield et al., Exp. Neurol. 175: 35-48, 2002; Giasson et al., Neuron34: 521-533, 2002; Lee et al., Proc Natl Acad. Sci. 99: 8968-8973,2002). The mutant mouse can carry a human SNCA gene that expresses anA53T, A30P, or E46K mutation. A treated mouse model can be observed fora decrease in symptoms associated with PD.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., RNA molecules,(double-stranded; single-stranded) that mediate RNAi. The iRNA agentspreferably mediate RNAi with respect to an endogenous SNCA gene of asubject

Generally, the iRNA agents of the instant invention include a region ofsufficient complementarity to an SNCA RNA, and are of sufficient lengthin terms of nucleotides, such that the iRNA agent, or a fragmentthereof, can mediate down regulation of the SNCA gene. It is notnecessary that there be perfect complementarity between the iRNA agentand the target, but the correspondence must be sufficient to enable theiRNA agent, or a cleavage product thereof, to direct sequence specificsilencing, e.g., by RNAi cleavage of an SNCA RNA.

Therefore, the iRNA agents featured in the instant invention includeagents comprising a sense strand and antisense strand each comprising asequence of at least 16, 17 or 18 nucleotides which is essentiallyidentical, as defined below, to a sequence included in FIG. 1, includingan SNCA sequence of Table 1, except that not more than 1, 2 or 3nucleotides per strand, respectively, have been substituted by othernucleotides (e.g., adenosine replaced by uracil), while essentiallyretaining the ability to inhibit SNCA expression in a mammalian cell.These agents will therefore possess at least 15 nucleotides identical toa sequence of FIG. 1, but 1, 2 or 3 base mismatches with respect toeither the target SNCA mRNA sequence or between the sense and antisensestrand are introduced. Mismatches to the target SNCA mRNA sequence,particularly in the antisense strand, are most tolerated in the terminalregions and if present are preferably in a terminal region or regions,e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus,most preferably within 6, 5, 4, or 3 nucleotides of the 5′-terminus ofthe sense strand or the 3′-terminus of the antisense strand. The sensestrand need only be sufficiently complementary with the antisense strandto maintain the over all double strand character of the molecule.

Single stranded regions of an iRNA agent will often be modified orinclude nucleoside surrogates, e.g., the unpaired region or regions of ahairpin structure, e.g., a region which links two complementary regions,can have modifications or nucleoside surrogates. Modifications tostabilize one or both of the 3′- or 5′-terminus of an iRNA agent, e.g.,against exonucleases, or to favor the antisense sRNA agent to enter intoRISC are also favored. Modifications can include C3 (or C6, C7, C12)amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers(C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol),special biotin or fluorescein reagents that come as phosphoramidites andthat have another DMT-protected hydroxyl group, allowing multiplecouplings during RNA synthesis. As discussed elsewhere herein, an iRNAagent will often be modified or include a ribose replacement monomersubunit (RRMS) in addition to the nucleotide surrogate. An RRMS replacesa ribose sugar on a ribonucleotide with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier. RRMS' are described ingreater detail below.

Although, in mammalian cells, long ds iRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and host. The iRNA agents of the presentinvention include molecules which are sufficiently short that they donot trigger the interferon response in mammalian cells. Thus, theadministration of a composition of an iRNA agent (e.g., formulated asdescribed herein) to a mammalian cell can be used to silence expressionof the SNCA gene while circumventing the interferon response. Moleculesthat are short enough that they do not trigger an interferon responseare termed sRNA agents or shorter iRNA agents herein. “sRNA agent orshorter iRNA agent” as used herein, refers to an iRNA agent, e.g., adouble stranded RNA agent or single strand agent, that is sufficientlyshort that it does not induce a deleterious interferon response in ahuman cell, e.g., it has a duplexed region of less than 60 butpreferably less than 50, 40, or 30 nucleotide pairs.

In addition to homology to target RNA and the ability to down regulate atarget gene, an iRNA agent will preferably have one or more of thefollowing properties:

-   -   (1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA        Agent section below;    -   (2) if single stranded it will have a 5′ modification which        includes one or more phosphate groups or one or more analogs of        a phosphate group;    -   (3) it will, despite modifications, even to a very large number,        or all of the nucleosides, have an antisense strand that can        present bases (or modified bases) in the proper three        dimensional framework so as to be able to form correct base        pairing and form a duplex structure with a homologous target RNA        which is sufficient to allow down regulation of the target,        e.g., by cleavage of the target RNA;    -   (4) it will, despite modifications, even to a very large number,        or all of the nucleosides, still have “RNA-like” properties,        i.e., it will possess the overall structural, chemical and        physical properties of an RNA molecule, even though not        exclusively, or even partly, of ribonucleotide-based content.        For example, an iRNA agent can contain, e.g., a sense and/or an        antisense strand in which all of the nucleotide sugars contain        e.g., 2′ fluoro in place of 2′ hydroxyl. This        deoxyribonucleotide-containing agent can still be expected to        exhibit RNA-like properties. While not wishing to be bound by        theory, the electronegative fluorine prefers an axial        orientation when attached to the C2′ position of ribose. This        spatial preference of fluorine can, in turn, force the sugars to        adopt a C_(3′)-endo pucker. This is the same puckering mode as        observed in RNA molecules and gives rise to the        RNA-characteristic A-family-type helix. Further, since fluorine        is a good hydrogen bond acceptor, it can participate in the same        hydrogen bonding interactions with water molecules that are        known to stabilize RNA structures. (Generally, it is preferred        that a modified moiety at the 2′ sugar position will be able to        enter into H-bonding which is more characteristic of the OH        moiety of a ribonucleotide than the H moiety of a        deoxyribonucleotide. A preferred iRNA agent will: exhibit a        C_(3′)-endo pucker in all, or at least 50, 75,80, 85, 90, or 95%        of its sugars; exhibit a C_(3′)-endo pucker in a sufficient        amount of its sugars that it can give rise to a the        RNA-characteristic A-family-type helix; will have no more than        20, 10, 5, 4, 3, 2, or 1 sugar which is not a C_(3′)-endo pucker        structure. These limitations are particularly preferably in the        antisense strand;    -   (5) regardless of the nature of the modification, and even        though the RNA agent can contain deoxynucleotides or modified        deoxynucleotides, particularly in overhang or other single        strand regions, it is preferred that DNA molecules, or any        molecule in which more than 50, 60, or 70% of the nucleotides in        the molecule, or more than 50, 60, or 70% of the nucleotides in        a duplexed region are deoxyribonucleotides, or modified        deoxyribonucleotides which are deoxy at the 2′ position, are        excluded from the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpanhandle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a double stranded iRNA.)

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”) asused herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interchain hybridization can form aregion of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides inlength. It should be equal to or less than 60, 50, 40, or 30,nucleotides in length. Preferred ranges are 15 to 30, 17 to 25, 19 to23, and 19 to 21 nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to orat least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 50 nucleotides inlength. It should be equal to or less than 60, 50, 40, or 30,nucleotides in length. Preferred ranges are 15 to 30,17 to 25, 19 to 23,and 19 to 21 nucleotides in length.

The double strand portion of a double stranded iRNA agent should beequal to or at least, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29,40, or 50 nucleotide pairs in length. It should be equal to or less than60, 50, 40, or 30, nucleotides pairs in length. Preferred ranges are 15to 30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length.

It may be desirable to modify one or both of the antisense and sensestrands of a double strand iRNA agent. In some cases they will have thesame modification or the same class of modification but in other casesthe sense and antisense strand will have different modifications, e.g.,in some cases it is desirable to modify only the sense strand. It may bedesirable to modify only the sense strand, e.g., to inactivate it, e.g.,the sense strand can be modified in order to inactivate the sense strandand prevent formation of an active sRNA/protein or RISC. This can beaccomplished by a modification which prevents 5′-phosphorylation of thesense strand, e.g., by modification with a 5′-O-methyl ribonucleotide(see Nykänen et al., (2001) ATP requirements and small interfering RNAstructure in the RNA interference pathway. Cell 107, 309-321.) Othermodifications which prevent phosphorylation can also be used, e.g.,simply substituting the 5′-OH by H rather than O-Me. Alternatively, alarge bulky group may be added to the 5′-phosphate turning it into aphosphodiester linkage, though this may be less desirable asphosphodiesterases can cleave such a linkage and release a functionalsRNA 5′-end. Antisense strand modifications include 5′ phosphorylationas well as any of the other 5′ modifications discussed herein,particularly the 5′ modifications discussed above in the section onsingle stranded iRNA molecules.

It is preferred that the sense and antisense strands be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains sense andantisense strands, preferably paired to contain an overhang, e.g., oneor two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3nucleotides. Most embodiments will have a 3′ overhang. Preferred iRNAagents will have single-stranded overhangs, preferably 3′ overhangs, of1 to 4, or preferably 2 or 3 nucleotides in length at each end. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. 5′ endsare preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe iRNA agent range discussed above. iRNA agents can resemble in lengthand structure the natural Dicer processed products from long dsRNAs.Embodiments in which the two strands of the sRNA agent are linked, e.g.,covalently linked are also included. Hairpin, or other single strandstructures which provide the required double stranded region, andpreferably a 3′ overhang are also within the invention.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a cell containingand/or secreting a certain product of the target gene when not incontact with the agent, will contain and/or secret at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar cell which has notbeen contacted with the agent. Such product of the target gene can, forexample, be a messenger RNA (mRNA), a protein, or a regulatory element.While not wishing to be bound by theory, it is believed that silencingby the agents described herein uses the RNAi machinery or process and aguide RNA, e.g., an iRNA agent of 15 to 30 nucleotide pairs.

As used herein, the term “complementary” is used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule, e.g., an SNCA mRNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA (e.g., a target SCNA mRNA) if the iRNAagent reduces the production of a protein encoded by the target RNA in acell. The iRNA agent may also be “exactly complementary” (excluding theRRMS containing subunit(s) to the target RNA, e.g., the target RNA andthe iRNA agent anneal, preferably to form a hybrid made exclusively ofWatson-Crick basepairs in the region of exact complementarity. A“sufficiently complementary” target RNA can include an internal region(e.g., of at least 10 nucleotides) that is exactly complementary to atarget SNCA RNA. Moreover, in some embodiments, the iRNA agentspecifically discriminates a single-nucleotide difference. In this case,the iRNA agent only mediates RNAi if exact complementary is found in theregion (e.g., within 7 nucleotides of) the single-nucleotide difference.Preferred iRNA agents will be based on or consist of or comprise theSNCA sense and antisense sequences provided in Table 1 and/or sequencesillustrated in FIG. 1.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Nucleic Acids Res. 22:2183-2196. Such rare or unusual RNAs, often termed modified RNAs(apparently because the are typically the result of a posttranscriptionally modification) are within the term unmodified RNA, asused herein. Modified RNA as used herein refers to a molecule in whichone or more of the components of the nucleic acid, namely sugars, bases,and phosphate moieties, are different from that which occur in nature,preferably different from that which occurs in the human body. Whilethey are referred to as modified “RNAs,” they will of course, because ofthe modification, include molecules which are not RNAs. Nucleosidesurrogates are molecules in which the ribophosphate backbone is replacedwith a non-ribophosphate construct that allows the bases to thepresented in the correct spatial relationship such that hybridization issubstantially similar to what is seen with a ribophosphate backbone,e.g., non-charged mimics of the ribophosphate backbone. Examples of allof the above are discussed herein.

Much of the discussion below refers to single strand molecules. In manyembodiments of the invention a ds iRNA agent, e.g., a partially doublestranded iRNA agent, is required or preferred. Thus, it is understoodthat double stranded structures (e.g. where two separate molecules arecontacted to form the double stranded region or where the doublestranded region is formed by intramolecular pairing (e.g., a hairpinstructure)) made of the single stranded structures described below arewithin the invention. Preferred lengths are described elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or the non-linking O of a phosphate moiety. In some cases themodification will occur at all of the subject positions in the nucleicacid but in many, and in fact in most, cases it will not. By way ofexample, a modification may only occur at a 3′ or 5′ terminal position,may only occur in a terminal region, e.g. at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of an RNA or may only occur in a single strand region of an RNA.E.g., a phosphorothioate modification at a non-linking O position mayonly occur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. Similarly, a modification mayoccur on the sense strand, antisense strand, or both. In some cases, thesense and antisense strand will have the same modifications or the sameclass of modifications, but in other cases the sense and antisensestrand will have different modifications, e.g., in some cases it may bedesirable to modify only one strand, e.g. the sense strand. In someembodiments it is particularly preferred, e.g., to enhance stability, toinclude particular bases in overhangs, or to include modifiednucleotides or nucleotide surrogates, in single strand overhangs, e.g.,in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to includepurine nucleotides in overhangs. In some embodiments all or some of thebases in a 3′ or 5′ overhang will be modified, e.g., with a modificationdescribed herein. Modifications can include, e.g., the use ofmodifications at the 2′ OH group of the ribose sugar, e.g., the use ofdeoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides,and modifications in the phosphate group, e.g., phosphothioatemodifications. Overhangs need not be homologous with the targetsequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of aribonucleic acid. The basic components are the ribose sugar, the base,the terminal phosphates, and phosphate internucleotide linkers. Wherethe bases are naturally occurring bases, e.g., adenine, uracil, guanineor cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (asdepicted) and W, X, Y, and Z are all O, Formula 1 represents a naturallyoccurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the above RNA components can confer improved properties, and,e.g., can render oligoribonucleotides more stable to nucleases.Unmodified oligoribonucleotides may also be less than optimal in termsof offering tethering points for attaching ligands or other moieties toan iRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

(i) alteration, e.g., replacement, of one or both of the non-linking (Xand Y) phosphate oxygens and/or of one or more of the linking (W and Z)phosphate oxygens (When the phosphate is in the terminal position, oneof the positions W or Z will not link the phosphate to an additionalelement in a naturally occurring ribonucleic acid. However, forsimplicity of terminology, except where otherwise noted, the W positionat the 5′ end of a nucleic acid and the terminal Z position at the 3′end of a nucleic acid, are within the term “linking phosphate oxygens”as used herein);

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesalereplacement of the ribose sugar with a structure other than ribose,e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with“dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone(bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule.

It is understood that the actual electronic structure of some chemicalentities cannot be adequately represented by only one canonical form(i.e. Lewis structure). While not wishing to be bound by theory, theactual structure can instead be some hybrid or weighted average of twoor more canonical forms, known collectively as resonance forms orstructures. Resonance structures are not discrete chemical entities andexist only on paper. They differ from one another only in the placementor “localization” of the bonding and nonbonding electrons for aparticular chemical entity. It can be possible for one resonancestructure to contribute to a greater extent to the hybrid than theothers. Thus, the written and graphical descriptions of the embodimentsof the present invention are made in terms of what the art recognizes asthe predominant resonance form for a particular species. For example,any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)would be represented by X=O and Y=N in the above figure.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors (cf. Bracket I in Formula 1 above). While not wishing to bebound by theory, it is believed that since the charged phosphodiestergroup is the reaction center in nucleolytic degradation, its replacementwith neutral structural mimics should impart enhanced nucleasestability. Again, while not wishing to be bound by theory, it can bedesirable, in some embodiment, to introduce alterations in which thecharged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates (see Bracket II of Formula 1 above).While not wishing to be bound by theory, it is believed that the absenceof a repetitively charged backbone diminishes binding to proteins thatrecognize polyanions (e.g. nucleases). Again, while not wishing to bebound by theory, it can be desirable in some embodiment, to introducealterations in which the bases are tethered by a neutral surrogatebackbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g.,n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogs. E.g., in preferred embodiments iRNA agents, especiallyantisense strands, are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorescein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Evaluation of iRNA Agents

One can evaluate a candidate iRNA agent, e.g., a modified iRNA agent. Ageneral approach is described below, but methods more specific to SNCAiRNA agents are discussed elsewhere herein. In general, one can test fora selected property by exposing the agent or modified molecule and acontrol molecule to the appropriate conditions and evaluating for thepresence of the selected property. For example, resistance to adegradent can be evaluated as follows. A candidate modified RNA (andpreferably a control molecule, usually the unmodified form) can beexposed to degradative conditions, e.g., exposed to a milieu, whichincludes a degradative agent, e.g., a nuclease. E.g., one can use abiological sample, e.g., one that is similar to a milieu, which might beencountered, in therapeutic use, e.g., blood or a cellular fraction,e.g., a cell-free homogenate or disrupted cells. The candidate andcontrol could then be evaluated for resistance to degradation by any ofa number of approaches. For example, the candidate and control could belabeled, preferably prior to exposure, with, e.g., a radioactive orenzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control andmodified RNA's can be incubated with the degradative agent, andoptionally a control, e.g., an inactivated, e.g., heat inactivated,degradative agent. A physical parameter, e.g., size, of the modified andcontrol molecules are then determined. They can be determined by aphysical method, e.g., by polyacrylamide gel electrophoresis or a sizingcolumn, to assess whether the molecule has maintained its originallength, or assessed functionally. Alternatively, Northern blot analysiscan be used to assay the length of an unlabeled modified molecule.

A functional assay can also be used to evaluate the candidate agent. Afunctional assay can be applied initially or after an earliernon-functional assay, (e.g., assay for resistance to degradation) todetermine if the modification alters the ability of the molecule tosilence gene expression. For example, a cell, e.g., a mammalian cell,such as a mouse or human cell, can be co-transfected with a plasmidexpressing a fluorescent protein, e.g., GFP, and a candidate RNA agenthomologous to the transcript encoding the fluorescent protein (see,e.g., WO 00/44914). For example, a modified siRNA homologous to the GFPmRNA can be assayed for the ability to inhibit GFP expression bymonitoring for a decrease in cell fluorescence, as compared to a controlcell, in which the transfection did not include the candidate siRNA,e.g., controls with no agent added and/or controls with a non-modifiedRNA added. Efficacy of the candidate agent on gene expression can beassessed by comparing cell fluorescence in the presence of the modifiedand unmodified iRNA agents.

The effect of the modified agent on target RNA levels can be verified byNorthern blot to assay for a decrease in the level of target mRNA, or byWestern blot to assay for a decrease in the level of target protein, ascompared to a negative control. Controls can include cells in which withno agent is added and/or cells in which a non-modified RNA is added.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R¹, R², and R³ are each, independently, H,(i.e. abasic nucleotides), adenine, guanine, cytosine and uracil,inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine,isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ togethercombine with R⁷ to form an [—O—CH₂—] covalently bound bridge between thesugar 2′ and 4′ carbons.

; H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

(a preferred A1, especially with regard to anti-sense strands, is chosenfrom 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′),5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether-methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-)).

; H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n) OR¹⁰, (CH₂)_(n) SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰;O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰;O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

X¹, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃ ⁻,H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each ofwhich may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH,(CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰,O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰,O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent describedherein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂—]covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5dyes); sulfur, silicon, boron or ester protecting group; intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (cholesterol, cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabeledmarkers, enzymes, haptens (e.g. biotin), transport/absorptionfacilitators (e.g., aspirin, vitamin E, folic acid), syntheticribonucleases (e.g., imidazole, bisimidazole, histamine, imidazoleclusters, acridine-imidazole conjugates, Eu3+ complexes oftetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20.Q is a spacer selected from the group consisting of abasic sugar, amide,carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea,sulfonamide, or morpholino, biotin or fluorescein reagents.

Preferred RNA agents in which the entire phosphate group has beenreplaced have the following structure (see Formula 3 below):

Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent,in which L is a linker, wherein one or both L may be present or absentand is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g);O(CH₂)_(g); S(CH₂)_(g). G is a functional group selected from the groupconsisting of siloxane, carbonate, carboxymethyl, carbamate, amide,thioether, ethylene oxide linker, sulfonate, sulfonamide,thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides),adenine, guanine, cytosine and uracil, inosine, thymine, xanthine,hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂R⁹; NHC(O)R⁸; cyano; mercapto, SR⁷;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridgebetween the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent describedherein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an[—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid. m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (seeFormula 4 below):SLR¹⁰⁰-(M-SLR²⁰⁰)_(x)-M-SLR³⁰⁰

FORMULA 4

S is a nucleoside surrogate selected from the group consisting ofmophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is alinker and is selected from the group consisting of CH₂(CH₂)_(g);N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may be absent. Mis an amide bond; sulfonamide; sulfinate; phosphate group; modifiedphosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasicnucleotides), adenine, guanine, cytosine and uracil, inosine, thymine,xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4,-triazoles, 2-pyridinones,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent describedherein; and g is 0-2.

Nuclease Resistant Monomers

An RNA, e.g., an iRNA agent, can incorporate a nuclease resistantmonomer (NRM). For example, the invention includes an iRNA agentdescribed herein, e.g., a palindromic iRNA agent, an iRNA agent having anon canonical pairing, an iRNA agent which targets a gene describedherein, e.g., an SNCA gene, an iRNA agent having an architecture orstructure described herein, an iRNA associated with an amphipathicdelivery agent described herein, an iRNA associated with a drug deliverymodule described herein, an iRNA agent administered as described herein,or an iRNA agent formulated as described herein, which also incorporatesan NRM.

An iRNA agent can include monomers which have been modified so as toinhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These monomers arereferred to herein as NRMs, or nuclease resistance promoting monomers ormodifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC(RNA-induced Silencing Complex), or the ability of the firstand second sequences to form a duplex with one another or to form aduplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an iRNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. Preferred modifications arethose that increase exonuclease and endonuclease resistance and thusprolong the half-life of the iRNA agent prior to interaction with theRISC complex, but at the same time do not render the iRNA agentresistant to endonuclease activity in the RISC complex. Again, while notwishing to be bound by any theory, it is believed that placement of themodifications at or near the 3′ and/or 5′ end of antisense strands canresult in iRNA agents that meet the preferred nuclease resistancecriteria delineated above. Again, still while not wishing to be bound byany theory, it is believed that placement of the modifications at e.g.,the middle of a sense strand can result in iRNA agents that arerelatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. An iRNA agent may include a duplex comprising a hybridizedsense and antisense strand, in which the antisense strand and/or thesense strand may include one or more of the modifications describedherein. The antisense strand may include modifications at the 3′ endand/or the 5′ end and/or at one or more positions that occur 1-6 (e.g.,1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sensestrand may include modifications at the 3′ end and/or the 5′ end and/orat any one of the intervening positions between the two ends of thestrand. The iRNA agent may also include a duplex comprising twohybridized antisense strands. The first and/or the second antisensestrand may include one or more of the modifications described herein.Thus, one and/or both antisense strands may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet thepreferred nuclease resistance criteria delineated above can include oneor more of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone:

(i) chiral (Sp) thioates. Thus, preferred NRMs include nucleotide dimerswith an enriched or pure for a particular chiral form of a modifiedphosphate group containing a heteroatom at the nonbridging position,e.g., Sp or Rp, at the position X, where this is the position normallyoccupied by the oxygen. The atom at X can also be S, Se, Nr₂, or Br₃.When X is S, enriched or chirally pure Sp linkage is preferred. Enrichedmeans at least 70, 80, 90, 95, or 99% of the preferred form. Such NRMsare discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base,and/or the phosphorus atom of a phosphate or modified phosphate backbonemoiety. Thus, preferred NRMs include monomers at the terminal positionderivatized at a cationic group. As the 5′ end of an antisense sequenceshould have a terminal —OH or phosphate group this NRM is preferably notused at the 5′ end of an anti-sense sequence. The group should beattached at a position on the base which minimizes interference with Hbond formation and hybridization, e.g., away form the face whichinteracts with the complementary base on the other strand, e.g, at the5′ position of a pyrimidine or a 7-position of a purine. These arediscussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRMs includeNon-phosphate linkages, e.g., a linkage of 4 atoms which confers greaterresistance to cleavage than does a phosphate bond. Examples include 3′CH2-NCH₃—O—CH₂-5′ and 3′ CH₂—NH—(O═)—CH₂-5′;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus,preferred NRM's can included these structures;

(v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides. Thus, otherpreferred NRM's include: L nucleosides and dimeric nucleotides derivedfrom L-nucleosides; 2′-5′ phosphate, non-phosphate and modifiedphosphate linkages (e.g., thiophosphates, phosphoramidates andboronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′linkages; monomers having an alpha linkage at the 1′ site on the sugar,e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include, e.g., atargeting moiety or a conjugated ligand described herein conjugated withthe monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasicmonomer, e.g., an abasic monomer as described herein (e.g., anucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclicaromatic monomer as described herein; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM'sinclude monomers, preferably at the terminal position, e.g., the 5′position, in which one or more atoms of the phosphate group isderivatized with a protecting group, which protecting group or groups,are removed as a result of the action of a component in the subject'sbody, e.g, a carboxyesterase or an enzyme present in the subject's body.E.g., a phosphate prodrug in which a carboxy esterase cleaves theprotected molecule resulting in the production of a thioate anion whichattacks a carbon adjacent to the O of a phosphate and resulting in theproduction of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent. As some NRMsinterfere with hybridization the total number incorporated, should besuch that acceptable levels of iRNA agent duplex formation aremaintained.

In some embodiments NRM modifications are introduced into the terminalcleavage site or in the cleavage region of a sequence (a sense strand orsequence) which does not target a desired sequence or gene in thesubject. This can reduce off-target silencing.

Chiral S_(P) Thioates

A modification can include the alteration, e.g., replacement, of one orboth of the non-linking (X and Y) phosphate oxygens- and/or of one ormore of the linking (W and Z) phosphate oxygens. Formula X below depictsa phosphate moiety linking two sugar/sugar surrogate-base moieties, SB₁and SB₂.

In certain embodiments, one of the non-linking phosphate oxygens in thephosphate backbone moiety (X and Y) can be replaced by any one of thefollowing: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., analkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl,etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodifiedphosphate group is achiral. However, replacement of one of thenon-linking oxygens with one of the above atoms or groups of atomsrenders the phosphorus atom chiral; in other words a phosphorus atom ina phosphate group modified in this way is a stereogenic center. Thestereogenic phosphorus atom can possess either the “R” configuration(herein R_(P)) or the “S” configuration (herein Sp). Thus if 60% of apopulation of stereogenic phosphorus atoms have the R_(P) configuration,then the remaining 40% of the population of stereogenic phosphorus atomshave the S_(P) configuration.

In some embodiments, iRNA agents, having phosphate groups in which aphosphate non-linking oxygen has been replaced by another atom or groupof atoms, may contain a population of stereogenic phosphorus atoms inwhich at least about 50% of these atoms (e.g., at least about 60% ofthese atoms, at least about 70% of these atoms, at least about 80% ofthese atoms, at least about 90% of these atoms, at least about 95% ofthese atoms, at least about 98% of these atoms, at least about 99% ofthese atoms) have the S_(P) configuration. Alternatively, iRNA agentshaving phosphate groups in which a phosphate non-linking oxygen has beenreplaced by another atom or group of atoms may contain a population ofstereogenic phosphorus atoms in which at least about 50% of these atoms(e.g., at least about 60% of these atoms, at least about 70% of theseatoms, at least about 80% of these atoms, at least about 90% of theseatoms, at least about 95% of these atoms, at least about 98% of theseatoms, at least about 99% of these atoms) have the R_(P) configuration.In other embodiments, the population of stereogenic phosphorus atoms mayhave the S_(P) configuration and may be substantially free ofstereogenic phosphorus atoms having the R_(P) configuration. In stillother embodiments, the population of stereogenic phosphorus atoms mayhave the R_(P) configuration and may be substantially free ofstereogenic phosphorus atoms having the S_(P) configuration. As usedherein, the phrase “substantially free of stereogenic phosphorus atomshaving the R_(P) configuration” means that moieties containingstereogenic phosphorus atoms having the R_(P) configuration cannot bedetected by conventional methods known in the art (chiral HPLC, ¹H NMRanalysis using chiral shift reagents, etc.). As used herein, the phrase“substantially free of stereogenic phosphorus atoms having the S_(P)configuration” means that moieties containing stereogenic phosphorusatoms having the S_(P) configuration cannot be detected by conventionalmethods known in the art (chiral HPLC, ¹H NMR analysis using chiralshift reagents, etc.).

In a preferred embodiment, modified iRNA agents contain aphosphorothioate group, i.e., a phosphate groups in which a phosphatenon-linking oxygen has been replaced by a sulfur atom. In an especiallypreferred embodiment, the population of phosphorothioate stereogenicphosphorus atoms may have the S_(P) configuration and be substantiallyfree of stereogenic phosphorus atoms having the R_(P) configuration.

Phosphorothioates may be incorporated into iRNA agents using dimerse.g., formulas X-1 and X-2. The former can be used to introducephosphorothioate

at the 3′ end of a strand, while the latter can be used to introducethis modification at the 5′ end or at a position that occurs e.g., 1, 2,3, 4, 5, or 6 nucleotides from either end of the strand. In the aboveformulas, Y can be 2-cyanoethoxy, W and Z can be O, R_(2′) can be, e.g.,a substituent that can impart the C-3 endo configuration to the sugar(e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and “BASE” can be anatural, unusual, or a universal base.

X-1 and X-2 can be prepared using chiral reagents or directing groupsthat can result in phosphorothioate-containing dimers having apopulation of stereogenic phosphorus atoms having essentially only theR_(P) configuration (i.e., being substantially free of the S_(P)configuration) or only the S_(P) configuration (i.e., beingsubstantially free of the R_(P) configuration). Alternatively, dimerscan be prepared having a population of stereogenic phosphorus atoms inwhich about 50% of the atoms have the R_(P) configuration and about 50%of the atoms have the S_(P) configuration. Dimers having stereogenicphosphorus atoms with the R_(P) configuration can be identified andseparated from dimers having stereogenic phosphorus atoms with the S_(P)configuration using e.g., enzymatic degradation and/or conventionalchromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groupsto the sugar, base, and/or the phosphorus atom of a phosphate ormodified phosphate backbone moiety. A cationic group can be attached toany atom capable of substitution on a natural, unusual or universalbase. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphatelinkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphatelinkages which can replace the phosphate group include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include the methylphosphonate and hydroxylamino groups.

3′-bridging Thiophosphates and 5′-bridging Thiophosphates; Locked-RNA,2′-5′ Linkages, Inverted Linkages, α-nucleosides; Conjugate Groups;Abasic Linkages; and 5′-phosphonates and 5′-phosphate Prodrugs

Referring to formula X above, modifications can include replacement ofone of the bridging or linking phosphate oxygens in the phosphatebackbone moiety (W and Z). Unlike the situation where only one of X or Yis altered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of iRNA agents containing a stereogenicphosphorus atom.

Modifications can also include linking two sugars via a phosphate ormodified phosphate group through the 2′ position of a first sugar andthe 5′ position of a second sugar. Also contemplated are invertedlinkages in which both a first and second sugar are each linked throughthe respective3′ positions. Modified RNA's can also include “abasic”sugars, which lack a nucleobase at C-1′. The sugar group can alsocontain one or more carbons that possess the opposite stereochemicalconfiguration than that of the corresponding carbon in ribose. Thus, amodified iRNA agent can include nucleotides containing e.g., arabinose,as the sugar. In another subset of this modification, the natural,unusual, or universal base may have the α-configuration. Modificationscan also include L-RNA.

Modifications can also include 5′-phosphonates, e.g.,P(O)(O⁻)₂—X—C^(5′)-sugar (X=CH2, CF2, CHF and 5′-phosphate prodrugs,e.g., P(O)[OCH2CH2SC(O)R]₂CH₂C^(5′)-sugar. In the latter case, theprodrug groups may be decomposed via reaction first with carboxyesterases. The remaining ethyl thiolate group via intramolecular S_(N)2displacement can depart as episulfide to afford the underivatizedphosphate group.

Modification can also include the addition of conjugating groupsdescribed elsewhere herein, which are preferably attached to an iRNAagent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed onlyat the terminus and others which can go at any position. Generally themodifications that can inhibit hybridization so it is preferably to usethem only in terminal regions, and preferable to not use them at thecleavage site or in the cleavage region of an sequence which targets asubject sequence or gene. The can be used anywhere in a sense sequence,provided that sufficient hybridization between the two sequences of theiRNA agent is maintained. In some embodiments it is desirable to put theNRM at the cleavage site or in the cleavage region of a sequence whichdoes not target a subject sequence or gene, as it can minimizeoff-target silencing.

In addition, an iRNA agent described herein can have an overhang whichdoes not form a duplex structure with the other sequence of the iRNAagent—it is an overhang, but it does hybridize, either with itself, orwith another nucleic acid, other than the other sequence of the iRNAagent.

In most cases, the nuclease-resistance promoting modifications will bedistributed differently depending on whether the sequence will target asequence in the subject (often referred to as an anti-sense sequence) orwill not target a sequence in the subject (often referred to as a sensesequence). If a sequence is to target a sequence in the subject,modifications which interfere with or inhibit endonuclease cleavageshould not be inserted in the region which is subject to RISC mediatedcleavage, e.g., the cleavage site or the cleavage region (As describedin Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated byreference). Cleavage of the target occurs about in the middle of a 20 or21 nt guide RNA, or about 10 or 11 nucleotides upstream of the firstnucleotide which is complementary to the guide sequence. As used hereincleavage site refers to the nucleotide on either side of the cleavagesite, on the target or on the iRNA agent strand which hybridizes to it.Cleavage region means an nucleotide with 1, 2, or 3 nucleotides of thecleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

An iRNA agent can have a first and a second strand chosen from thefollowing:

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which does not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

An iRNA agent can also target two sequences and can have a first andsecond strand chosen from:

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which targets a sequence and which preferably does nothave an NRM modification at the cleavage site or in the cleavage region;

a first strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand) and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1,2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

Ribose Mimics

An RNA, e.g., an iRNA agent, can incorporate a ribose mimic. Inaddition, the invention includes iRNA agents having a ribose mimic andanother element described herein. E.g., the invention includes an iRNAagent described herein, e.g., a palindromic iRNA agent, an iRNA agenthaving a non canonical pairing, an iRNA agent which targets a genedescribed herein, e.g., an SNCA gene, an iRNA agent having anarchitecture or structure described herein, an iRNA associated with anamphipathic delivery agent described herein, an iRNA associated with adrug delivery module described herein, an iRNA agent administered asdescribed herein, or an iRNA agent formulated as described herein, whichalso incorporates a ribose mimic.

Thus, an aspect of the invention features an iRNA agent that includes asecondary hydroxyl group, which can increase efficacy and/or confernuclease resistance to the agent. Nucleases, e.g., cellular nucleases,can hydrolyze nucleic acid phosphodiester bonds, resulting in partial orcomplete degradation of the nucleic acid. The secondary hydroxy groupconfers nuclease resistance to an iRNA agent by rendering the iRNA agentless prone to nuclease degradation relative to an iRNA which lacks themodification. While not wishing to be bound by theory, it is believedthat the presence of a secondary hydroxyl group on the iRNA agent canact as a structural mimic of a 3′ ribose hydroxyl group, thereby causingit to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attachedto a carbon atom substituted by two other carbons and a hydrogen. Thesecondary hydroxyl group that confers nuclease resistance as describedabove can be part of any acyclic carbon-containing group. The hydroxylmay also be part of any cyclic carbon-containing group, and preferablyone or more of the following conditions is met (1) there is no ribosemoiety between the hydroxyl group and the terminal phosphate group or(2) the hydroxyl group is not on a sugar moiety which is coupled to abase. The hydroxyl group is located at least two bonds (e.g., at leastthree bonds away, at least four bonds away, at least five bonds away, atleast six bonds away, at least seven bonds away, at least eight bondsaway, at least nine bonds away, at least ten bonds away, etc.) from theterminal phosphate group phosphorus of the iRNA agent. In preferredembodiments, there are five intervening bonds between the terminalphosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bondsbetween the terminal phosphate group phosphorus and the secondaryhydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agentdescribed herein. The iRNA agent may be connected directly or indirectly(e.g., through a spacer or linker) to “W” of the phosphate group. Thesespacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—,—(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6),abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,disulfide, thiourea, sulfonamide, or morpholino, or biotin andfluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified(e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group,W and Z can be independently NH, O, or S; and X and Y can beindependently S, Se, BH₃ ⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxyor amino (including alkylamino, arylamino, etc.). Preferably, W, X and Zare O and Y is S.

R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl,optionally substituted with hydroxyl, amino, halo, phosphate or sulfateand/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken togetherwith R₄ or R₆ to form a ring of 5-12 atoms.

R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken togetherwith R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken togetherwith R₄ to form a ring of 5-12 atoms.

R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken togetherwith R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_(q)C(O)NHR₉; T is hydrogenor a functional group; n and q are each independently 1-100; R₈ isC₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C1-C10 alkyl, C6-C10aryl or a solid support agent.

Preferred embodiments may include one of more of the following subsetsof iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, A can be connecteddirectly or indirectly through a terminal 3′ or 5′ ribose sugar carbonof the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O andY is S.

In still yet another subset of RNAi agent delivery modules, n is 1, andR₂ and R₆ are taken together to form a ring containing six atoms and R₄and R₅ are taken together to form a ring containing six atoms.Preferably, the ring system is a trans-decalin. For example, the RNAiagent delivery module of this subset can include a compound of Formula(Y-1):

The functional group can be, for example, a targeting group (e.g., asteroid or a carbohydrate), a reporter group (e.g., a fluorophore), or alabel (an isotopically labeled moiety). The targeting group can furtherinclude protein binding agents, endothelial cell targeting groups (e.g.,RGD peptides and mimetics), cancer cell targeting groups (e.g., folateVitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates,polyglutamates, polyaspartates), multivalent mannose (for e.g.,macrophage testing), lactose, galactose, N-acetyl-galactosamine,monoclonal antibodies, glycoproteins, lectins, melanotropin, orthyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. The synthesized compounds can be separatedfrom a reaction mixture and further purified by a method such as columnchromatography, high pressure liquid chromatography, orrecrystallization. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Ribose Replacement Monomer Subunits

iRNA agents can be modified in a number of ways which can optimize oneor more characteristics of the iRNA agent. An RNA agent, e.g., an iRNAagent can include a ribose replacement monomer subunit (RRMS), such asthose described herein In addition, an iRNA agent can have an RRMS andanother element described herein. E.g., the invention includes an iRNAagent described herein, e.g., a palindromic iRNA agent, an iRNA agenthaving a non canonical pairing, an iRNA agent which targets a genedescribed herein, e.g., an SNCA gene, an iRNA agent having anarchitecture or structure described herein, an iRNA associated with anamphipathic delivery agent described herein, an iRNA associated with adrug delivery module described herein, an iRNA agent administered asdescribed herein, or an iRNA agent formulated as described herein, whichalso incorporates a RRMS.

The ribose sugar of one or more ribonucleotide subunits of an iRNA agentcan be replaced with another moiety, e.g., a non-carbohydrate(preferably cyclic) carrier. A ribonucleotide subunit in which theribose sugar of the subunit has been so replaced is referred to hereinas an RRMS. A cyclic carrier may be a carbocyclic ring system, i.e., allring atoms are carbon atoms, or a heterocyclic ring system, i.e., one ormore ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. Thecyclic carrier may be a monocyclic ring system, or may contain two ormore rings, e.g. fused rings. The cyclic carrier may be a fullysaturated ring system, or it may contain one or more double bonds.

The carriers further include (i) at least two “backbone attachmentpoints” and (ii) at least one “tethering attachment point.” A “backboneattachment point” as used herein refers to a functional group, e.g. ahydroxyl group, or generally, a bond available for, and that is suitablefor incorporation of the carrier into the backbone, e.g., the phosphate,or modified phosphate, e.g., sulfur containing, backbone, of aribonucleic acid. A “tethering attachment point” as used herein refersto a constituent ring atom of the cyclic carrier, e.g., a carbon atom ora heteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., aligand, e.g., a targeting or delivery moiety, or a moiety which alters aphysical property, e.g., lipophilicity, of an iRNA agent. Optionally,the selected moiety is connected by an intervening tether to the cycliccarrier. Thus, it will include a functional group, e.g., an amino group,or generally, provide a bond, that is suitable for incorporation ortethering of another chemical entity, e.g., a ligand to the constituentring.

Incorporation of one or more RRMSs described herein into an RNA agent,e.g., an iRNA agent, particularly when tethered to an appropriateentity, can confer one or more new properties to the RNA agent and/oralter, enhance or modulate one or more existing properties in the RNAmolecule. E.g., it can alter one or more of lipophilicity or nucleaseresistance. Incorporation of one or more RRMSs described herein into aniRNA agent can, particularly when the RRMS is tethered to an appropriateentity, modulate, e.g., increase, binding affinity of an iRNA agent to atarget mRNA, change the geometry of the duplex form of the iRNA agent,alter distribution or target the iRNA agent to a particular part of thebody, or modify the interaction with nucleic acid binding proteins(e.g., during RISC formation and strand separation).

Accordingly, in one aspect, the invention features, an iRNA agentpreferably comprising a first strand and a second strand, wherein atleast one subunit having a formula (R-1) is incorporated into at leastone of said strands.

Referring to formula (R-1), X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, 0, S,CR⁹R¹⁰, or absent; and Z is CR¹¹R¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a),OR^(b), (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b), provided that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^(a) or OR^(b) and that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b)(when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ willinclude R^(a) and one will include R^(b); when the RRMS is internal, twoof R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R); further providedthat preferably OR^(a) may only be present with (CH₂)_(n)OR^(b) and(CH₂)_(n)OR^(a) may only be present with OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is C₁-C₂₀ alkyl substituted with NR^(c)R^(d); R⁸ is C₁-C₆ alkyl; R¹³is hydroxy, C₁-C₄ alkoxy, or halo; and R¹⁴ is NR^(c)R⁷.

Each of A and C is, independently, O or S.

B is OH, O⁻, or

R^(c) is H or C1-C6 alkyl; R^(d) is H or a ligand; and n is 1-4.

In a preferred embodiment the ribose is replaced with a pyrrolinescaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

In other preferred embodiments the ribose is replaced with a piperidinescaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a piperazinescaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a morpholinoscaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a decalinscaffold, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹together are C⁶ cycloalkyl.

In other preferred embodiments the ribose is replaced with adecalin/indane scaffold and, and X is CH₂; Y is CR⁹R¹⁰; and Z isCR¹¹R¹²; and R⁵ and R¹¹ together are C⁵ cycloalkyl.

In other preferred embodiments, the ribose is replaced with ahydroxyproline scaffold.

RRMSs described herein may be incorporated into any double-strandedRNA-like molecule described herein, e.g., an iRNA agent. An iRNA agentmay include a duplex comprising a hybridized sense and antisense strand,in which the antisense strand and/or the sense strand may include one ormore of the RRMSs described herein. An RRMS can be introduced at one ormore points in one or both strands of a double-stranded iRNA agent. AnRRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or5′ end of the sense strand or at near (within 2 or 3 positions of) the3′ end of the antisense strand. In some embodiments it is preferred tonot have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ endof the antisense strand. An RRMS can be internal, and will preferably bepositioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand. In an embodiment, aniRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the3′ end of the antisense strand and at (or within 1, 2, or 3 positionsof) the 3′ end of the sense strand. In an embodiment, an iRNA agent mayhave an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of theantisense strand and an RRMS at the 5′ end of the sense strand, in whichboth ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one oneach strand and are hydrophobic moieties. While not wishing to be boundby theory, it is believed that pairing of the hydrophobic ligands canstabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′end of the sense strand, in which both RRMSs may share the same ligand(e.g., cholic acid) via connection of their individual tethers toseparate positions on the ligand. A ligand shared between two proximalRRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end ofthe sense strand and an RRMS at an internal position of the sensestrand. An iRNA agent may have an RRMS at an internal position of thesense strand; or may have an RRMS at an internal position of theantisense strand; or may have an RRMS at an internal position of thesense strand and an RRMS at an internal position of the antisensestrand.

In preferred embodiments the iRNA agent includes a first and secondsequence, which are preferably two separate molecules as opposed to twosequences located on the same strand, have sufficient complementarity toeach other to hybridize (and thereby form a duplex region), e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains first andsecond sequences, preferable paired to contain an overhang, e.g., one ortwo 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides.Most embodiments will have a 3′ overhang. Preferred sRNA agents willhave single-stranded overhangs, preferably 3′ overhangs, of 1 orpreferably 2 or 3 nucleotides in length at each end. The overhangs canbe the result of one strand being longer than the other, or the resultof two strands of the same length being staggered. 5′ ends arepreferably phosphorylated.

Tethered Entities

A wide variety of entities can be tethered to an iRNA agent, e.g., tothe carrier of an RRMS. Examples are described below in the context ofan RRMS but that is only preferred, entities can be coupled at otherpoints to an iRNA agent. Preferred entities are those which target to aneural cell, e.g., a neural cell expressing SNCA.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe RRMS carrier. In preferred embodiments, the ligand is attached tothe carrier via an intervening tether. As discussed above, the ligand ortethered ligand may be present on the RRMS monomer when the RRMS monomeris incorporated into the growing strand. In some embodiments, the ligandmay be incorporated into a “precursor” RRMS after a “precursor” RRMSmonomer has been incorporated into the growing strand. For example, anRRMS monomer having, e.g., an amino-terminated tether (i.e., having noassociated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporated into agrowing sense or antisense strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor RRMS bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor RRMS tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g, molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. For example, in a preferredembodiment, a ligand will provide enhanced selectivity to a neural cell,such as in the brain. Preferred ligands will not take part in duplexpairing in a duplexed nucleic acid.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,insulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a neural cell.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP. In oneembodiment, a ligand can facilitate the movement of the iRNA agentacross the blood-brain barrier.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a neuralcell. Ligands may also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalentmannose, or multivalent fucose.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-liver target tissue ofthe body. Preferably, the target tissue is the brain. Other moleculesthat can bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the liver and therefore less likely to be cleared from thebody.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 300, 35, 40, 45, or 50 amino acids long (see Table 3,for example).

TABLE 3 Exemplary Cell Permeation Peptides Cell Permeation Peptide Aminoacid Sequence Reference Penetratin RQIKIWFQNRRMKWKK Derossi et al., (SEQID NO:31) J. Biol. Chem. 269:10444, 1994 Tat fragment GRKKRRQRRRPPQCVives et al., (48-60) (SEQ ID NO:32) J. Biol. Chem., 272:16010, 1997Signal GALFLGWLGAAGSTMGAWS Chaloin et al., Sequence- QPKKKRKV Biochem.Biophys. based peptide (SEQ ID NO:33) Res. Commun., 243:601, 1998 PVECLLIILRRRIRKQAHAHSK Elmquist et al., (SEQ ID NO:34) Exp. Cell Res.,269:237, 2001 Transportan GWTLNSAGYLLKINLKALA Pooga et al., ALAKKILFASEB

(SEQ ID NO:35) J., 12:67, 1998 Amphiphilic KLALKLALKALKAALKLA Oehlke etal., Mol. model peptide (SEQ ID NO:36) Ther., 2:339, 2000 Arg₉ RRRRRRRRRMitchell et al., J. (SEQ ID NO:37) Pept. Res., 56:3 18, 2000 Bacterialcell KFFKFFKFFK wall (SEQ ID NO:38) permeating LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID NO:39) Cecropin P1SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (SEQ ID NO:40) α-defensinACYCRIPACIAGERRYGTCIYQGRLWAFCC (SEQ ID NO:41) b-defensinDHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO:42) BactenecinRKCRIVVIRVCR (SEQ ID NO:43) PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (SEQ ID NO:44) Indolicidin ILPWKWPWWPWRR-NH2 (SEQ IDNO:45)

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Thepeptide moiety can be an L-peptide or D-peptide. In another alternative,the peptide moiety can include a hydrophobic membrane translocationsequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGFhaving the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:46). An RFGFanalogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:47)containing a hydrophobic MTS can also be a targeting moiety. The peptidemoiety can be a “delivery” peptide, which can carry large polarmolecules including peptides, oligonucleotides, and protein across cellmembranes. For example, sequences from the HIV Tat protein(GRKKRRQRRRPPQ (SEQ ID NO:48) and the Drosophila Antennapedia protein(RQIKIWFQNRRMKWKK (SEQ ID NO:49) have been found to be capable offunctioning as delivery peptides. A peptide or peptidomimetic can beencoded by a random sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary (Lam et al., Nature 354:82-84, 1991). Preferably the peptide orpeptidomimetic tethered to an iRNA agent via an incorporated monomerunit is a cell targeting peptide such as an arginine-glycine-asparticacid (RGD)-peptide, or RGD mimic. A peptide moiety can range in lengthfrom about 5 amino acids to about 40 amino acids. The peptide moietiescan have a structural modification, such as to increase stability ordirect conformational properties. Any of the structural modificationsdescribed below can be utilized.

A “cell permeation peptide” is capable of permeating a cell, e.g.,a—mammalian cell, such as a human cell. A cell permeation peptide canalso include a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an RRMS can be anamphipathic α-helical peptide. Exemplary amphipathic α-helical peptidesinclude, but are not limited to, cecropins, lycotoxins, paradaxins,buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins,S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

Methods for Making iRNA Agents

iRNA agents can include modified or non-naturally occurring bases, e.g.,bases described herein. In addition, iRNA agents can have a modified ornon-naturally occurring base and another element described herein. E.g.,the invention includes an iRNA agent described herein, e.g., apalindromic iRNA agent, an iRNA agent having a non canonical pairing, aniRNA agent which targets a gene described herein, e.g., an SNCA gene, aniRNA agent having an architecture or structure described herein, an iRNAassociated with an amphipathic delivery agent described herein, an iRNAassociated with a drug delivery module described herein, an iRNA agentadministered as described herein, or an iRNA agent formulated asdescribed herein, which also incorporates a modified or non-naturallyoccurring base.

The synthesis and purification of oligonucleotide peptide conjugates canbe performed by established methods. See, for example, Trufert et al.,Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugatesin Antisense Technology,” in Antisense Drug Technology, ed. S. T.Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified tocreate a constrained peptide that adopts a distinct and specificpreferred conformation, which can increase the potency and selectivityof the peptide. For example, the constrained peptide can be anazapeptide (Gante, Synthesis 405-413, 1989). An azapeptide issynthesized by replacing the α-carbon of an amino acid with a nitrogenatom without changing the structure of the amino acid side chain. Forexample, the azapeptide can be synthesized by using hydrazine intraditional peptide synthesis coupling methods, such as by reactinghydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be an N-methylpeptide. N-methyl peptides are composed of N-methyl amino acids, whichprovide an additional methyl group in the peptide backbone, therebypotentially providing additional means of resistance to proteolyticcleavage. N-methyl peptides can by synthesized by methods known in theart (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99,2000; Cell Penetrating Peptides: Processes and Applications, Langel,ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate.Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002).For example, an Ant or Tat peptide can be an N-methyl peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be a β-peptide.β-peptides form stable secondary structures such as helices, pleatedsheets, turns and hairpins in solutions. Their cyclic derivatives canfold into nanotubes in the solid state. β-peptides are resistant todegradation by proteolytic enzymes. β-peptides can be synthesized bymethods known in the art. For example, an Ant or Tat peptide can be aβ-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate.Oligocarbamate peptides are internalized into a cell by a transportpathway facilitated by carbamate transporters. For example, an Ant orTat peptide can be an oligocarbamate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be an oligoureaconjugate (or an oligothiourea conjugate), in which the amide bond of apeptidomimetic is replaced with a urea moiety. Replacement of the amidebond provides increased resistance to degradation by proteolyticenzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In oneembodiment, an oligourea conjugate is tethered to an iRNA agent for usein oral delivery. The backbone in each repeating unit of an oligoureapeptidomimetic can be extended by one carbon atom in comparison with thenatural amino acid. The single carbon atom extension can increasepeptide stability and lipophilicity, for example. An oligourea peptidecan therefore be advantageous when an iRNA agent is directed for passagethrough a bacterial cell wall, or when an iRNA agent must traverse theblood-brain barrier, such as for the treatment of a neurologicaldisorder. In one embodiment, a hydrogen bonding unit is conjugated tothe oligourea peptide, such as to create an increased affinity with areceptor. For example, an Ant or Tat peptide can be an oligoureaconjugate (or an oligothiourea conjugate).

The dsRNA peptide conjugates of the invention can be affiliated with,e.g., tethered to, RRMSs occurring at various positions on an iRNAagent. For example, a peptide can be terminally conjugated, on eitherthe sense or the antisense strand, or a peptide can be bisconjugated(one peptide tethered to each end, one conjugated to the sense strand,and one conjugated to the antisense strand). In another option, thepeptide can be internally conjugated, such as in the loop of a shorthairpin iRNA agent. In yet another option, the peptide can be affiliatedwith a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, whichcan encapsulate one or more iRNA agents (such as for delivery to abiological system and/or a cell), and a peptide moiety tethered to theoutside of the carrier molecule, such as for targeting the carriercomplex to a particular tissue or cell type. A carrier complex can carryadditional targeting molecules on the exterior of the complex, orfusogenic agents to aid in cell delivery. The one or more iRNA agentsencapsulated within the carrier can be conjugated to lipophilicmolecules, which can aid in the delivery of the agents to the interiorof the carrier.

A carrier molecule or structure can be, for example, a micelle, aliposome (e.g., a cationic liposome), a nanoparticle, a microsphere, ora biodegradable polymer. A peptide moiety can be tethered to the carriermolecule by a variety of linkages, such as a disulfide linkage, an acidlabile linkage, a peptide-based linkage, an oxyamino linkage or ahydrazine linkage. For example, a peptide-based linkage can be a GFLGpeptide. Certain linkages will have particular advantages, and theadvantages (or disadvantages) can be considered depending on the tissuetarget or intended use. For example, peptide based linkages are stablein the blood stream but are susceptible to enzymatic cleavage in thelysosomes.

Definitions

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp² carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The term “alkoxy” refers to an —O-alkyl radical. The term “aminoalkyl”refers to an alkyl substituted with an amino. The term “mercapto” refersto an —SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalentspecies of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom capable of substitutioncan be substituted by a substituent. Examples of aryl moieties include,but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom capable of substitution can besubstituted by a substituent. The cycloalkyl groups herein described mayalso contain fused rings. Fused rings are rings that share a commoncarbon-carbon bond. Examples of cycloalkyl moieties include, but are notlimited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom capable of substitution can be substituted by asubstituent. The heterocyclyl groups herein described may also containfused rings. Fused rings are rings that share a common carbon-carbonbond. Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcapable of substitution can be substituted by a substituent.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” andthe like refer to radicals of adenine, cytosine, guanine, thymine, anduracil.

As used herein, an “unusual” nucleobase can include any one of thefollowing:

2-methyladeninyl,

N6-methyladeninyl,

2-methylthio-N-6-methyladeninyl,

N6-isopentenyladeninyl,

2-methylthio-N-6-isopentenyladeninyl,

N6-(cis-hydroxyisopentenyl)adeninyl,

2-methylthio-N-6-(cis-hydroxyisopentenyl)adeninyl,

N6-glycinylcarbamoyladeninyl,

N6-threonylcarbamoyladeninyl,

2-methylthio-N-6-threonyl carbamoyladeninyl,

N6-methyl-N-6-threonylcarbamoyladeninyl,

N6-hydroxynorvalylcarbamoyladeninyl,

2-methylthio-N-6-hydroxynorvalyl carbamoyladeninyl,

N6,N6-dimethyladeninyl,

3-methylcytosinyl,

5-methylcytosinyl,

2-thiocytosinyl,

5-formylcytosinyl,

N4-methylcytosinyl,

5-hydroxymethylcytosinyl,

1-methylguaninyl,

N2-methylguaninyl,

7-methylguaninyl,

N2,N2-dimethylguaninyl,

N2,N2,7-trimethylguaninyl,

1-methylguaninyl,

7-cyano-7-deazaguaninyl,

7-aminomethyl-7-deazaguaninyl,

pseudouracilyl,

dihydrouracilyl,

5-methyluracilyl,

1-methylpseudouracilyl,

2-thiouracilyl,

4-thiouracilyl,

2-thiothyminyl

5-methyl-2-thiouracilyl,

3-(3-amino-3-carboxypropyl)uracilyl,

5-hydroxyuracilyl,

5-methoxyuracilyl,

uracilyl 5-oxyacetic acid,

uracilyl 5-oxyacetic acid methyl ester,

5-(carboxyhydroxymethyl)uracilyl,

5-(carboxyhydroxymethyl)uracilyl methyl ester,

5-methoxycarbonylmethyluracilyl,

5-methoxycarbonylmethyl-2-thiouracilyl,

5-aminomethyl-2-thiouracilyl,

5-methylaminomethyluracilyl,

5-methylaminomethyl-2-thiouracilyl,

5-methylaminomethyl-2-selenouracilyl,

5-carbamoylmethyluracilyl,

5-carboxymethylaminomethyluracilyl,

5-carboxymethylaminomethyl-2-thiouracilyl,

3-methyluracilyl,

1-methyl-3-(3-amino-3-carboxypropyl)pseudouracilyl,

5-carboxymethyluracilyl,

5-methyldihydrouracilyl, or

3-methylpseudouracilyl.

Palindromes

An RNA, e.g., an iRNA agent, can have a palindrome structure asdescribed herein. For example, the iRNA agents of the invention cantarget more than one RNA region. For example, an iRNA agent can includea first and second sequence that are sufficiently complementary to eachother to hybridize. The first sequence can be complementary to a firsttarget sequence of an SNCA RNA and the second sequence can becomplementary to a second target sequence of an SNCA RNA. The first andsecond target sequences can differ by at least 1 nucleotide. The firstand second sequences of the iRNA agent can be on different RNA strands,and the mismatch between the first and second sequences can be less than50%, 40%, 30%, 20%, 10%, 5%, or 1%. The first and second sequences ofthe iRNA agent can be on the same RNA strand, and in a relatedembodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNAagent can be in bimolecular form. The first and second sequences of theiRNA agent can be fully complementary to each other.

The first and second target RNA regions can be on transcripts encoded byfirst and second sequence variants, e.g., first and second alleles, ofan SNCA gene. The sequence variants can be mutations, or polymorphisms,for example. The first target RNA region can include a nucleotidesubstitution, insertion, or deletion relative to the second target RNAregion, or the second target RNA region can a mutant or variant of thefirst target region.

The compositions of the invention can include mixtures of iRNA agentmolecules. For example, one iRNA agent can contain a first sequence anda second sequence sufficiently complementary to each other to hybridize,and in addition the first sequence is complementary to a first targetRNA region and the second sequence is complementary to a second targetRNA region. The mixture can also include at least one additional iRNAagent variety that includes a third sequence and a fourth sequencesufficiently complementary to each other to hybridize, and where thethird sequence is complementary to a third target RNA region and thefourth sequence is complementary to a fourth target RNA region. Inaddition, the first or second sequence can be sufficiently complementaryto the third or fourth sequence to be capable of hybridizing to eachother. The first and second sequences can be on the same or differentRNA strands, and the third and fourth sequences can be on the same ordifferent RNA strands.

An iRNA agent can include a first sequence complementary to a firstvariant SNCA RNA target region and a second sequence complementary to asecond variant SNCA RNA target region. The first and second varianttarget RNA regions can include allelic variants, mutations (e.g., pointmutations), or polymorphisms of the SNCA target gene. Other thanCanonical Watson-Crick Duplex Structures.

Other than Canonical Watson-Crick Duplex Structures

An RNA, e.g., an iRNA agent can include monomers that can form otherthan a canonical Watson-Crick pairing with another monomer, e.g., amonomer on another strand. The use of “other than canonical Watson-Crickpairing” between monomers of a duplex can be used to control, often topromote, melting of all or part of a duplex. The iRNA agent can includea monomer at a selected or constrained position that results in a firstlevel of stability in the iRNA agent duplex (e.g., between the twoseparate molecules of a double stranded iRNA agent) and a second levelof stability in a duplex between a sequence of an iRNA agent and anothersequence molecule, e.g., a target or off-target sequence in a subject.In some cases the second duplex has a relatively greater level ofstability, e.g., in a duplex between an anti-sense sequence of an iRNAagent and a target mRNA. In this case one or more of the monomers, theposition of the monomers in the iRNA agent, and the target sequence(sometimes referred to herein as the selection or constraintparameters), are selected such that the iRNA agent duplex has acomparatively lower free energy of association (which while not wishingto be bound by mechanism or theory, is believed to contribute toefficacy by promoting disassociation of the duplex iRNA agent in thecontext of the RISC) while the duplex formed between an antisensetargeting sequence and its target sequence, has a relatively higher freeenergy of association (which while not wishing to be bound by mechanismor theory, is believed to contribute to efficacy by promotingassociation of the antisense sequence and the target RNA).

In other cases the second duplex has a relatively lower level ofstability, e.g., in a duplex between a sense sequence of an iRNA agentand an off-target mRNA. In this case one or more of the monomers, theposition of the monomers in the iRNA agent, and an off-target sequence,are selected such that the iRNA agent duplex is has a comparativelyhigher free energy of association while the duplex formed between asense targeting sequence and its off-target sequence, has a relativelylower free energy of association (which while not wishing to be bound bymechanism or theory, is believed to reduce the level of off-targetsilencing by promoting disassociation of the duplex formed by the sensestrand and the off-target sequence).

Thus, inherent in the structure of the iRNA agent is the property ofhaving a first stability for the intra-iRNA agent duplex and a secondstability for a duplex formed between a sequence from the iRNA agent andanother RNA, e.g., a target mRNA. As discussed above, this can beaccomplished by judicious selection of one or more of the monomers at aselected or constrained position, the selection of the position in theduplex to place the selected or constrained position, and selection ofthe sequence of a target sequence (e.g., the particular region of atarget gene which is to be targeted). The iRNA agent sequences whichsatisfy these requirements are sometimes referred to herein asconstrained sequences. Exercise of the constraint or selectionparameters can be, e.g., by inspection or by computer assisted methods.Exercise of the parameters can result in selection of a target sequenceand of particular monomers to give a desired result in terms of thestability, or relative stability, of a duplex.

Thus, in another aspect, the invention features an iRNA agent whichincludes: a first sequence which targets a first target region and asecond sequence which targets a second target region. The first andsecond sequences have sufficient complementarity to each other tohybridize, e.g., under physiological conditions, e.g., underphysiological conditions but not in contact with a helicase or otherunwinding enzyme. In a duplex region of the iRNA agent, at a selected orconstrained position, the first target region has a first monomer, andthe second target region has a second monomer. The first and secondmonomers occupy complementary or corresponding positions. One, andpreferably both monomers are selected such that the stability of thepairing of the monomers contribute to a duplex between the first andsecond sequence will differ form the stability of the pairing betweenthe first or second sequence with a target sequence.

Usually, the monomers will be selected (selection of the target sequencemay be required as well) such that they form a pairing in the iRNA agentduplex which has a lower free energy of dissociation, and a lower Tm,than will be possessed by the paring of the monomer with itscomplementary monomer in a duplex between the iRNA agent sequence and atarget RNA duplex.

The constraint placed upon the monomers can be applied at a selectedsite or at more than one selected site. By way of example, theconstraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positionsin the iRNA agent duplex. E.g., a constrained or selected site can bepresent within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of aduplexed sequence. A constrained or selected site can be present in themiddle of the duplex region, e.g., it can be more than 3, 4, 5, or 6,positions from the end of a duplexed region.

In some embodiment the duplex region of the iRNA agent will havemismatches, in addition to the selected or constrained site or sites.Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which donot form canonical Watson-Crick pairs or which do not hybridize.Overhangs are discussed in detail elsewhere herein but are preferablyabout 2 nucleotides in length. The overhangs can be complementary to thegene sequences being targeted or can be other sequence. TT is apreferred overhang sequence. The first and second iRNA agent sequencescan also be joined, e.g., by additional bases to form a hairpin, or byother non-base linkers.

The monomers can be selected such that: first and second monomers arenaturally occurring ribonucleotides, or modified ribonucleotides havingnaturally occurring bases, and when occupying complemetary sites eitherdo not pair and have no substantial level of H-bonding, or form a noncanonical Watson-Crick pairing and form a non-canonical pattern of Hbonding, which usually have a lower free energy of dissociation thanseen in a canonical Watson-Crick pairing, or otherwise pair to give afree energy of association which is less than that of a preselectedvalue or is less, e.g., than that of a canonical pairing. When one (orboth) of the iRNA agent sequences duplexes with a target, the first (orsecond) monomer forms a canonical Watson-Crick pairing with the base inthe complemetary position on the target, or forms a non-canonicalWatson-Crick pairing having a higher free energy of dissociation and ahigher Tm than seen in the pairing in the iRNA agent. The classicalWatson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonicalWatson-Crick pairings are known in the art and can include, U-U, G-G,G-A_(trans), G-A_(cis), and GU.

The monomer in one or both of the sequences is selected such that, itdoes not pair, or forms a pair with its corresponding monomer in theother sequence which minimizes stability (e.g., the H bonding formedbetween the monomer at the selected site in the one sequence and itsmonomer at the corresponding site in the other sequence are less stablethan the H bonds formed by the monomer one (or both) of the sequenceswith the respective target sequence. The monomer of one or both strandsis also chosen to promote stability in one or both of the duplexes madeby a strand and its target sequence. E.g., one or more of the monomersand the target sequences are selected such that at the selected orconstrained position, there is are no H bonds formed, or a non canonicalpairing is formed in the iRNA agent duplex, or they otherwise pair togive a free energy of association which is less than that of apreselected value or is less, e.g., than that of a canonical pairing,but when one (or both) sequences form a duplex with the respectivetarget, the pairing at the selected or constrained site is a canonicalWatson-Crick paring.

The inclusion of such a monomer will have one or more of the followingeffects: it will destabilize the iRNA agent duplex, it will destabilizeinteractions between the sense sequence and unintended target sequences,sometimes referred to as off-target sequences, and duplex interactionsbetween the a sequence and the intended target will not be destabilized.

A non-naturally occurring or modified monomer or monomers can be chosensuch that when a non-naturally occurring or modified monomer occupies aposition at the selected or constrained position in an iRNA agent theyexhibit a first free energy of dissociation and when one (or both) ofthem pairs with a naturally occurring monomer, the pair exhibits asecond free energy of dissociation, which is usually higher than that ofthe pairing of the first and second monomers. E.g., when the first andsecond monomers occupy complementary positions they either do not pairand have no substantial level of H-bonding, or form a weaker bond thanone of them would form with a naturally occurring monomer, and reducethe stability of that duplex, but when the duplex dissociates at leastone of the strands will form a duplex with a target in which theselected monomer will promote stability, e.g., the monomer will form amore stable pair with a naturally occurring monomer in the targetsequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thiopyrimidine analog of U or T.

When placed in complementary positions of the iRNA agent these monomerswill pair very poorly and will minimize stability. However, a duplex isformed between 2 amino A and the U of a naturally occurring target, or aduplex is between 2-thio U and the A of a naturally occurring target or2-thio T and the A of a naturally occurring target will have arelatively higher free energy of dissociation and be more stable.

The term “other than canonical Watson-Crick pairing” as used herein,refers to a pairing between a first monomer in a first sequence and asecond monomer at the corresponding position in a second sequence of aduplex in which one or more of the following is true: (1) there isessentially no pairing between the two, e.g., there is no significantlevel of H bonding between the monomers or binding between the monomersdoes not contribute in any significant way to the stability of theduplex; (2) the monomers are a non-canonical paring of monomers having anaturally occurring bases, i.e., they are other than A-T, A-U, or G-C,and they form monomer-monomer H bonds, although generally the H bondingpattern formed is less strong than the bonds formed by a canonicalpairing; or (3) at least one of the monomers includes a non-naturallyoccurring bases and the H bonds formed between the monomers is,preferably formed is less strong than the bonds formed by a canonicalpairing, namely one or more of A-T, A-U, G-C.

The term “off-target” as used herein, refers to as a sequence other thanthe sequence to be silenced.

Universal Bases: “wild-cards”; shape-based complementarity

Bi-stranded, multisite replication of a base pair betweendifluorotoluene and adenine: confirmation by ‘inverse’ sequencing. Liu,D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)

(Importance of terminal base pair hydrogen-bonding in 3′-endproofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.;Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

(Selective and stable DNA base pairing without hydrogen bonds. Matray,T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)

(Difluorotoluene, a nonpolar isostere for thymine, codes specificallyand efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.;Rumney IV, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)

(Structure and base pairing properties of a replicable nonpolar isosterefor deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org.Chem., 1998, 63, 9652-9656)

(Universal bases for hybridization, replication and chain termination.Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.;Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914)

-   (1. Efforts toward the expansion of the genetic alphabet:    Information storage and replication with unnatural hydrophobic base    pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.;    Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2.    Rational design of an unnatural base pair with increased kinetic    selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.;    Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)

(Efforts toward expansion of the genetic alphabet: replication of DNAwith three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.;Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)

(1. Efforts toward expansion of the genetic alphabet: Optimization ofinterbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.;McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000,122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNApolymerase recognition of a highly stable, self-pairing hydrophobicbase. McMinn, D. L.; Ogawa A. K.; Wu, Y.; Liu, J.; Schultz, P. G.;Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)

(A stable DNA duplex containing a non-hydrogen-bonding and non-shapecomplementary base couple: Interstrand stacking as the stabilitydetermining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew.Chem. Int. Ed., 2001, 40, 3012-3014)

(2,2′-Bipyridine Ligandoside: A novel building block for modifying DNAwith intra-duplex metal complexes. Weizman, H.; Tor, Y. J. Am. Chem.Soc., 2001, 123, 3375-3376)

(Minor groove hydration is critical to the stability of DNA duplexes.Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)

(Effect of the Universal base 3-nitropyrrole on the selectivity ofneighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W.Organic Lett., 2001, 3, 1977-1980. 2. Effect of the1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability ofDNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R.Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure anddeoxyribonucleic acid sequencing with a universal nucleosides:1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang,P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995,117, 1201-1209)

(

(Model studies directed toward a general triplex DNA recognition scheme:a novel DNA base that binds a CG base-pair in an organic solvent.Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)

(A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside.J. Org. Chem., 2001, 66, 2067-2071)

(Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine.Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122,2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into singleguanine bulge as evidenced by photooxidation of GC doublet, Nakatani,K.; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001,11, 335-337)

Other universal bases can have the following formulas:

wherein:

Q is N or CR⁴⁴;

Q′ is N or CR⁴⁵;

Q″ is N or CR⁴⁷;

Q′″ is N or CR⁴⁹;

Q^(iv) is N or CR⁵⁰;

R⁴⁴ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁵ forms —OCH₂O—;

R⁴⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁴ or R⁴⁶ forms —OCH₂O—;

R⁴⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁵ or R⁴⁷ forms —OCH₂O—;

R⁴⁷ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁶ or R⁴⁸ forms —OCH₂O—;

R⁴⁸ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁷ forms —OCH2O—;

R⁴⁹ R⁵⁰, R⁵¹, R⁵², R⁵³, R⁵⁴, R⁵⁷, R⁵⁸, R⁵⁹, R⁶⁰, R⁶¹, R⁶², R⁶³, R⁶⁴,R⁶⁵, R⁶⁶, R⁶⁷, R⁶⁸, R⁶⁹, R⁷⁰, R⁷¹, and R⁷² are each independentlyselected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂,NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o);

R⁵⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o), or when takentogether with R⁵⁶ forms a fused aromatic ring which may be optionallysubstituted;

R⁵⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o), or when takentogether with R⁵⁵ forms a fused aromatic ring which may be optionallysubstituted;

R¹⁷ is halo, NH₂, NHR^(b), or NR^(b)R^(c);

R^(b) is C₁-C₆ alkyl or a nitrogen protecting group;

R^(c) is C₁-C₆ alkyl; and

R^(o) is alkyl optionally substituted with halo, hydroxy, nitro,protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷,or NC(O)R^(o).

Examples of universal bases include:

Asymmetrical Modifications

An RNA, e.g., an iRNA agent, can be asymmetrically modified as describedherein, and as described in International Application Serial No.PCT/US04/07070, filed Mar. 8, 2004, which is hereby incorporated byreference.

In addition, the invention includes iRNA agents having asymmetricalmodifications and another element described herein. E.g., the inventionincludes an iRNA agent described herein, e.g., a palindromic iRNA agent,an iRNA agent having a non canonical pairing, an iRNA agent whichtargets a gene described herein, e.g., an SNCA gene, an iRNA agenthaving an architecture or structure described herein, an iRNA associatedwith an amphipathic delivery agent described herein, an iRNA associatedwith a drug delivery module described herein, an iRNA agent administeredas described herein, or an iRNA agent formulated as described herein,which also incorporates an asymmetrical modification.

An asymmetrically modified iRNA agent is one in which a strand has amodification which is not present on the other strand. An asymmetricalmodification is a modification found on one strand but not on the otherstrand. Any modification, e.g., any modification described herein, canbe present as an asymmetrical modification. An asymmetrical modificationcan confer any of the desired properties associated with a modification,e.g., those properties discussed herein. E.g., an asymmetricalmodification can: confer resistance to degradation, an alteration inhalf life; target the iRNA agent to a particular target, e.g., to aparticular tissue; modulate, e.g., increase or decrease, the affinity ofa strand for its complement or target sequence; or hinder or promotemodification of a terminal moiety, e.g., modification by a kinase orother enzymes involved in the RISC mechanism pathway. The designation ofa modification as having one property does not mean that it has no otherproperty, e.g., a modification referred to as one which promotesstabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanisticmodel, it is believed that asymmetrical modification allows an iRNAagent to be optimized in view of the different or “asymmetrical”functions of the sense and antisense strands. For example, both strandscan be modified to increase nuclease resistance, however, since somechanges can inhibit RISC activity, these changes can be chosen for thesense stand. In addition, since some modifications, e.g., targetingmoieties, can add large bulky groups that, e.g., can interfere with thecleavage activity of the RISC complex, such modifications are preferablyplaced on the sense strand. Thus, targeting moieties, especially bulkyones (e.g. cholesterol), are preferentially added to the sense strand.In one embodiment, an asymmetrical modification in which a phosphate ofthe backbone is substituted with S, e.g., a phosphorothioatemodification, is present in the antisense strand, and a 2′ modification,e.g., 2′ OMe is present in the sense strand. A targeting moiety can bepresent at either (or both) the 5′ or 3′ end of the sense strand of theiRNA agent. In a preferred example, a P of the backbone is replaced withS in the antisense strand, 2′OMe is present in the sense strand, and atargeting moiety is added to either the 5′ or 3′ end of the sense strandof the iRNA agent.

In a preferred embodiment an asymmetrically modified iRNA agent has amodification on the sense strand which modification is not found on theantisense strand and the antisense strand has a modification which isnot found on the sense strand.

Each strand can include one or more asymmetrical modifications. By wayof example: one strand can include a first asymmetrical modificationwhich confers a first property on the iRNA agent and the other strandcan have a second asymmetrical modification which confers a secondproperty on the iRNA. E.g., one strand, e.g., the sense strand can havea modification which targets the iRNA agent to a tissue, and the otherstrand, e.g., the antisense strand, has a modification which promoteshybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the sameproperty, e.g., to increase resistance to nucleolytic degradation, butdifferent modifications are chosen for the sense and the antisensestrands, e.g., because the modifications affect other properties aswell. E.g., since some changes can affect RISC activity thesemodifications are chosen for the sense strand.

In one embodiment, one strand has an asymmetrical 2′ modification, e.g.,a 2′ OMe modification, and the other strand has an asymmetricalmodification of the phosphate backbone, e.g., a phosphorothioatemodification. So, in one embodiment the antisense strand has anasymmetrical 2′ OMe modification and the sense strand has anasymmetrical phosphorothioate modification (or vice versa). In aparticularly preferred embodiment, the RNAi agent will have asymmetrical2′-O alkyl, preferably, 2′-OMe modifications on the sense strand andasymmetrical backbone P modification, preferably a phosphorothioatemodification in the antisense strand. There can be one or multiple2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunitsof the sense strand can be so modified. There can be one or multiplephosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of thesubunits of the antisense strand can be so modified. It is preferable tohave an iRNA agent wherein there are multiple 2′-OMe modifications onthe sense strand and multiple phophorothioate modifications on theantisense strand. All of the subunits on one or both strands can be somodified. A particularly preferred embodiment of multiple asymmetricmodifications on both strands has a duplex region about 20-21, andpreferably 19, subunits in length and one or two 3′ overhangs of about 2subunits in length.

Asymmetrical modifications are useful for promoting resistance todegradation by nucleases, e.g., endonucleases. iRNA agents can includeone or more asymmetrical modifications which promote resistance todegradation. In preferred embodiments the modification on the antisensestrand is one which will not interfere with silencing of the target,e.g., one which will not interfere with cleavage of the target. Most ifnot all sites on a strand are vulnerable, to some degree, to degradationby endonucleases. One can determine sites which are relativelyvulnerable and insert asymmetrical modifications which inhibitdegradation. It is often desirable to provide asymmetrical modificationof a UA site in an iRNA agent, and in some cases it is desirable toprovide the UA sequence on both strands with asymmetrical modification.Examples of modifications which inhibit endonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety on the U, especially ona sense strand; modification of the backbone, e.g., with the replacementof an O with an S, in the phosphate backbone, e.g., the provision of aphosphorothioate modification, on the U or the A or both, especially onan antisense strand; replacement of the U with a C5 amino linker;replacement of the A with a G (sequence changes are preferred to belocated on the sense strand and not the antisense strand); andmodification of the at the 2′, 6′, 7′, or 8′ position. Preferredembodiments are those in which one or more of these modifications arepresent on the sense but not the antisense strand, or embodiments wherethe antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation byexonucleases. Asymmetrical modifications can include those in which onlyone strand is modified as well as those in which both are modified. Inpreferred embodiments the modification on the antisense strand is onewhich will not interfere with silencing of the target, e.g., one whichwill not interfere with cleavage of the target. Some embodiments willhave an asymmetrical modification on the sense strand, e.g., in a 3′overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g.,in a 3′ overhang, e.g., at the 3′ terminus. If the modificationsintroduce moieties of different size it is preferable that the larger beon the sense strand. If the modifications introduce moieties ofdifferent charge it is preferable that the one with greater charge be onthe sense strand.

Examples of modifications which inhibit exonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g.,at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule orat the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicatedby the context); modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P in a 3′ overhang, e.g., atthe 3′ terminus; combination of a 2′ modification, e.g., provision of a2′ O Me moiety and modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrolidine in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxene, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus. Preferred embodiments are those in whichone or more of these modifications are present on the sense but not theantisense strand, or embodiments where the antisense strand has fewer ofsuch modifications.

Modifications, e.g., those described herein, which affect targeting canbe provided as asymmetrical modifications. Targeting modifications whichcan inhibit silencing, e.g., by inhibiting cleavage of a target, can beprovided as asymmetrical modifications of the sense strand. Abiodistribution altering moiety, e.g., cholesterol, can be provided inone or more, e.g., two, asymmetrical modifications of the sense strand.Targeting modifications which introduce moieties having a relativelylarge molecular weight, e.g., a molecular weight of more than 400, 500,or 1000 daltons, or which introduce a charged moiety (e.g., having morethan one positive charge or one negative charge) can be placed on thesense strand.

Modifications, e.g., those described herein, which modulate, e.g.,increase or decrease, the affinity of a strand for its compliment ortarget, can be provided as asymmetrical modifications. These include: 5methyl U; 5 methyl C; pseudouridine, Locked nucleic acids include: 2thio U and 2-amino-A. In some embodiments one or more of these isprovided on the antisense strand.

iRNA agents have a defined structure, with a sense strand and anantisense strand, and in many cases short single strand overhangs, e.g.,of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetricalmodification can be used to optimize the activity of such a structure,e.g., by being placed selectively within the iRNA. E.g., the end regionof the iRNA agent defined by the 5′ end of the sense strand and the 3′end of the antisense strand is important for function. This region caninclude the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang.In preferred embodiments asymmetrical modifications which result in oneor more of the following are used: modifications of the 5′ end of thesense strand which inhibit kinase activation of the sense strand,including, e.g., attachments of conjugates which target the molecule orthe use modifications which protect against 5′ exonucleolyticdegradation; or modifications of either strand, but preferably the sensestrand, which enhance binding between the sense and antisense strand andthereby promote a “tight” structure at this end of the molecule.

The end region of the iRNA agent defined by the 3′ end of the sensestrand and the 5′end of the antisense strand is also important forfunction. This region can include the terminal 2, 3, or 4 pairednucleotides and any 3′ overhang. Preferred embodiments includeasymmetrical modifications of either strand, but preferably the sensestrand, which decrease binding between the sense and antisense strandand thereby promote an “open” structure at this end of the molecule.Such modifications include placing conjugates which target the moleculeor modifications which promote nuclease resistance on the sense strandin this region. Modification of the antisense strand which inhibitkinase activation are avoided in preferred embodiments.

Exemplary modifications for asymmetrical placement in the sense strandinclude the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S); these modifications can be used topromote nuclease resistance;

(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminaland/or internal positions); these modifications can be used to promotenuclease resistance or to enhance binding of the sense to the antisensestrand, the 3′ modifications can be used at the 5′ end of the sensestrand to avoid sense strand activation by RISC;

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S)these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the antisense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the antisense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleicacids (HNA's) and cyclohexene nucleic acids (CeNA's)); thesemodifications can be used to promote nuclease resistance or to inhibitbinding of the sense to the antisense strand, or can be used at the 5′end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines), thesemodifications can be used to promote nuclease resistance or to enhancebinding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus),these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e.g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates;these modifications can be used to promote nuclease resistance or totarget the molecule, or can be used at the 5′ end of the sense strand toavoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisensestrand include the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S);

(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the3′ end); e.g., with P═O or P═S preferably at the 3′-end, thesemodifications are preferably excluded from the 5′ end region as they mayinterfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the3′-end, these modifications are preferably excluded from the 5′ endregion as they may interfere with kinase activity;

(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modificationsare preferably excluded from the 5′ end region as they may contribute tounwanted enhancements of paring between the sense and antisense strands,it is often preferred to have a “loose” structure in the 5′ region,additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);

cationic groups and Zwitterionic groups at 2′-position of sugar;3′-position of the sugar; as nucleobase modifications (e.g., C-5modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6modified purines);

conjugate groups (preferably at terminal positions), e.g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates,but bulky groups or generally groups which inhibit RISC activity shouldare less preferred.

The 5′-OH of the antisense strand should be kept free to promoteactivity. In some preferred embodiments modifications that promotenuclease resistance should be included at the 3′ end, particularly inthe 3′ overhang.

In another aspect, the invention features a method of optimizing, e.g.,stabilizing, an iRNA agent. The method includes selecting a sequencehaving activity, introducing one or more asymmetric modifications intothe sequence, wherein the introduction of the asymmetric modificationoptimizes a property of the iRNA agent but does not result in a decreasein activity.

The decrease in activity can be less than a preselected level ofdecrease. In preferred embodiments decrease in activity means a decreaseof less than 5, 10, 20, 40, or 50% activity, as compared with anotherwise similar iRNA lacking the introduced modification. Activitycan, e.g., be measured in vivo, or in vitro, with a result in eitherbeing sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and inparticular the properties discussed in the section on asymmetricalmodifications provided herein. The modification can be any asymmetricalmodification, e.g., an asymmetric modification described in the sectionon asymmetrical modifications described herein. Particularly preferredasymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMemodifications, particularly in the sense sequence, and modifications ofa backbone O, particularly phosphorothioate modifications, in theantisense sequence.

In a preferred embodiment a sense sequence is selected and provided withan asymmetrical modification, while in other embodiments an antisensesequence is selected and provided with an asymmetrical modification. Insome embodiments both sense and antisense sequences are selected andeach provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or bothof the sense and antisense sequence. A sequence can have at least 2, 4,6, 8, or more modifications and all or substantially all of the monomersof a sequence can be modified.

Z-X-Y Architecture

An RNA, e.g., an iRNA agent, can have a Z-X-Y architecture or structuresuch as those described herein. In addition, an iRNA agent can have aZ-X-Y structure and another element described herein. E.g., theinvention includes an iRNA agent described herein, e.g., a palindromiciRNA agent, an iRNA agent having a non canonical pairing, an iRNA agentwhich targets a gene described herein, e.g., an SNCA gene, an iRNAassociated with an amphipathic delivery agent described herein, an iRNAassociated with a drug delivery module described herein, an iRNA agentadministered as described herein, or an iRNA agent formulated asdescribed herein, which also incorporates a Z-X-Y architecture.

Thus, an iRNA agent can have a first segment, the Z region, a secondsegment, the X region, and optionally a third region, the Y region:

-   -   Z-X-Y.

It may be desirable to modify subunits in one or both of Z and/or Y onone hand and X on the other hand. In some cases they will have the samemodification or the same class of modification but it will more often bethe case that the modifications made in Z and/or Y will differ fromthose made in X.

The Z region typically includes a terminus of an iRNA agent. The lengthof the Z region can vary, but will typically be from 2-14, morepreferably 2-10, subunits in length. It typically is single stranded,i.e., it will not base pair with bases of another strand, though it mayin some embodiments self associate, e.g., to form a loop structure. Suchstructures can be formed by the end of a strand looping back and formingan intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairscan form, having a looped out or connecting region, typically of 2 ormore subunits which do not pair. This can occur at one or both ends of astrand. A typical embodiment of a Z region is a single strand overhang,e.g., an over hang of the length described elsewhere herein. The Zregion can thus be or include a 3′ or 5′ terminal single strand. It canbe sense or antisense strand but if it is antisense it is preferred thatit is a 3-overhang. Typical inter-subunit bonds in the Z region include:P═O; P═S; S—P═S; P—NR₂; and P—BR₂. Chiral P═X, where X is S, N, or B)inter-subunit bonds can also be present. Other preferred Z regionsubunit modifications (also discussed elsewhere herein) can include:3′-OR, 3′SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alphaconfiguration bases; and 2′ arabino modifications.

The X region will in most cases be duplexed, in the case of a singlestrand iRNA agent, with a corresponding region of the single strand, orin the case of a double stranded iRNA agent, with the correspondingregion of the other strand. The length of the X region can vary but willtypically be between 10-45 and more preferably between 15 and 35subunits. Particularly preferred region X's will include 17, 18, 19, 29,21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengthsare described elsewhere herein and can be used. Typical X regionsubunits include 2′-OH subunits. In typical embodiments phosphateinter-subunit bonds are preferred while phophorothioate or non-phosphatebonds are absent. Other modifications preferred in the X region include:modifications to improve binding, e.g., nucleobase modifications;cationic nucleobase modifications; and C-5 modified pyrimidines, e.g.,allylamines. Some embodiments have 4 or more consecutive 2′OH subunits.While the use of phosphorothioate is sometimes non preferred they can beused if they connect less than 4 consecutive 2′OH subunits.

The Y region will generally conform to the parameters set out for the Zregions. However, the X and Z regions need not be the same, differenttypes and numbers of modifications can be present, and in fact, one willusually be a 3′ overhang and one will usually be a 5′ overhang.

In a preferred embodiment the iRNA agent will have a Y and/or Z regioneach having ribonucleosides in which the 2′-OH is substituted, e.g.,with 2′-OMe or other alkyl; and an X region that includes at least fourconsecutive ribonucleoside subunits in which the 2′-OH remainsunsubstituted.

The subunit linkages (the linkages between subunits) of an iRNA agentcan be modified, e.g., to promote resistance to degradation. Numerousexamples of such modifications are disclosed herein, one example ofwhich is the phosphorothioate linkage. These modifications can beprovided between the subunits of any of the regions, Y, X, and Z.However, it is preferred that their occurrence is minimized and inparticular it is preferred that consecutive modified linkages beavoided.

In a preferred embodiment the iRNA agent will have a Y and Z region eachhaving ribonucleosides in which the 2′-OH is substituted, e.g., with2′-OMe; and an X region that includes at least four consecutivesubunits, e.g., ribonucleoside subunits in which the 2′-OH remainsunsubstituted.

As mentioned above, the subunit linkages of an iRNA agent can bemodified, e.g., to promote resistance to degradation. Thesemodifications can be provided between the subunits of any of theregions, Y, X, and Z. However, it is preferred that they are minimizedand in particular it is preferred that consecutive modified linkages beavoided.

Thus, in a preferred embodiment, not all of the subunit linkages of theiRNA agent are modified and more preferably the maximum number ofconsecutive subunits linked by other than a phosphodiester bond will be2, 3, or 4. Particularly preferred iRNA agents will not have four ormore consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, inwhich each subunit is joined by modified linkages—i.e. linkages thathave been modified to stabilize them from degradation as compared to thephosphodiester linkages that naturally occur in RNA and DNA.

It is particularly preferred to minimize the occurrence in region X.Thus, in preferred embodiments each of the nucleoside subunit linkagesin X will be phosphodiester linkages, or if subunit linkages in region Xare modified, such modifications will be minimized. E.g., although the Yand/or Z regions can include inter subunit linkages which have beenstabilized against degradation, such modifications will be minimized inthe X region, and in particular consecutive modifications will beminimized. Thus, in preferred embodiments the maximum number ofconsecutive subunits linked by other than a phospodiester bond will be2, 3, or 4. Particularly preferred X regions will not have four or moreconsecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, inwhich each subunits is joined by modified linkages—i.e., linkages thathave been modified to stabilize them from degradation as compared to thephosphodiester linkages that naturally occur in RNA and DNA.

In a preferred embodiment Y and/or Z will be free of phosphorothioatelinkages, though either or both may contain other modifications, e.g.,other modifications of the subunit linkages.

In a preferred embodiment, region X, or in some cases, the entire iRNAagent, has no more than 3 or no more than 4 subunits having identical 2′moieties.

In a preferred embodiment, region X, or in some cases, the entire iRNAagent, has no more than 3 or no more than 4 subunits having identicalsubunit linkages.

In a preferred embodiment, one or more phosphorothioate linkages (orother modifications of the subunit linkage) are present in Y and/or Z,but such modified linkages do not connect two adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g.,any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by alinkage other than a phosphorothioate linkage.

In a preferred embodiment, each of Y and/or Z independently has only onephosphorothioate linkage between adjacent subunits, e.g., nucleosides,having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is asecond set of adjacent subunits, e.g., nucleosides, having a 2′modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that secondset is connected by a linkage other than a phosphorothioate linkage,e.g., a modified linkage other than a phosphorothioate linkage.

In a preferred embodiment, each of Y and/or Z independently has morethan one phosphorothioate linkage connecting adjacent pairs of subunits,e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkylnucleosides, but at least one pair of adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, arebe connected by a linkage other than a phosphorothioate linkage, e.g., amodified linkage other than a phosphorothioate linkage.

In a preferred embodiment one of the above recited limitation onadjacent subunits in Y and or Z is combined with a limitation on thesubunits in X. E.g., one or more phosphorothioate linkages (or othermodifications of the subunit linkage) are present in Y and/or Z, butsuch modified linkages do not connect two adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g.,any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by alinkage other than a phosphorothioate linkage. In addition, the X regionhas no more than 3 or no more than 4 identical subunits, e.g., subunitshaving identical 2′ moieties or the X region has no more than 3 or nomore than 4 subunits having identical subunit linkages.

A Y and/or Z region can include at least one, and preferably 2, 3 or 4of a modification disclosed herein. Such modifications can be chosen,independently, from any modification described herein, e.g., fromnuclease resistant subunits, subunits with modified bases, subunits withmodified intersubunit linkages, subunits with modified sugars, andsubunits linked to another moiety, e.g., a targeting moiety. In apreferred embodiment more than 1 of such subunit can be present but insome embodiments it is preferred that no more than 1, 2, 3, or 4 of suchmodifications occur, or occur consecutively. In a preferred embodimentthe frequency of the modification will differ between Y and/or Z and X,e.g., the modification will be present one of Y and/or Z or X and absentin the other.

An X region can include at least one, and preferably 2, 3 or 4 of amodification disclosed herein. Such modifications can be chosen,independently, from any modification described herein, e.g., fromnuclease resistant subunits, subunits with modified bases, subunits withmodified intersubunit linkages, subunits with modified sugars, andsubunits linked to another moiety, e.g., a targeting moiety. In apreferred embodiment more than 1 of such subunits can b present but insome embodiments it is preferred that no more than 1, 2, 3, or 4 of suchmodifications occur, or occur consecutively.

An RRMS (described elsewhere herein) can be introduced at one or morepoints in one or both strands of a double-stranded iRNA agent. An RRMScan be placed in a Y and/or Z region, at or near (within 1, 2, or 3positions) of the 3′ or 5′ end of the sense strand or at near (within 2or 3 positions of) the 3′ end of the antisense strand. In someembodiments it is preferred to not have an RRMS at or near (within 1, 2,or 3 positions of) the 5′ end of the antisense strand. An RRMS can bepositioned in the X region, and will preferably be positioned in thesense strand or in an area of the antisense strand not critical forantisense binding to the target.

Differential Modification of Terminal Duplex Stability

In one aspect, the invention features an iRNA agent which can havedifferential modification of terminal duplex stability (DMTDS).

In addition, the invention includes iRNA agents having DMT/DS andanother element described herein. E.g., the invention includes an iRNAagent described herein, e.g., a palindromic iRNA agent, an iRNA agenthaving a non canonical pairing, an iRNA agent which targets a genedescribed herein, e.g., an SNCA gene, an iRNA agent having anarchitecture or structure described herein, an iRNA associated with anamphipathic delivery agent described herein, an iRNA associated with adrug delivery module described herein, an iRNA agent administered asdescribed herein, or an iRNA agent formulated as described herein, whichalso incorporates DMTDS.

iRNA agents can be optimized by increasing the propensity of the duplexto disassociate or melt (decreasing the free energy of duplexassociation), in the region of the 5′ end of the antisense strandduplex. This can be accomplished, e.g., by the inclusion of subunits,which increase the propensity of the duplex to disassociate or melt inthe region of the 5′ end of the antisense strand. This can also beaccomplished by the attachment of a ligand that increases the propensityof the duplex to disassociate of melt in the region of the 5′end. Whilenot wishing to be bound by theory, the effect may be due to promotingthe effect of an enzyme such as a helicase, for example, promoting theeffect of the enzyme in the proximity of the 5′ end of the antisensestrand.

The inventors have also discovered that iRNA agents can be optimized bydecreasing the propensity of the duplex to disassociate or melt(increasing the free energy of duplex association), in the region of the3′ end of the antisense strand duplex. This can be accomplished, e.g.,by the inclusion of subunits which decrease the propensity of the duplexto disassociate or melt in the region of the 3′ end of the antisensestrand. It can also be accomplished by the attachment of ligand thatdecreases the propensity of the duplex to disassociate or melt in theregion of the 5′end.

Modifications which increase the tendency of the 5′ end of the duplex todissociate can be used alone or in combination with other modificationsdescribed herein, e.g., with modifications which decrease the tendencyof the 3′ end of the duplex to dissociate. Likewise, modifications whichdecrease the tendency of the 3′ end of the duplex to dissociate can beused alone or in combination with other modifications described herein,e.g., with modifications which increase the tendency of the 5′ end ofthe duplex to dissociate.

Decreasing the Stability of the AS 5′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotedissociation or melting (e.g., on the free energy of association ordissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:

A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferredover G:C (I = inosine);

-   -   mismatches, e.g., non-canonical or other than canonical pairings        (as described elsewhere herein) are preferred over canonical        (A:T, A:U, G:C) pairings;    -   pairings which include a universal base are preferred over        canonical pairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5′ R₁ N₁ N₂ N₃ N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₂ 3′ AS 3′ R₃ N₁ N₂ N₃N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₄ 5′ S:AS P₁ P₂ P₃ P₄ P₅ [N] P⁻⁵ P⁻⁴ P⁻³P⁻² P⁻¹ 5′

S indicates the sense strand; AS indicates antisense strand; R₁indicates an optional (and nonpreferred) 5′ sense strand overhang; R₂indicates an optional (though preferred) 3′ sense overhang; R₃ indicatesan optional (though preferred) 3′ antisense sense overhang; R₄ indicatesan optional (and nonpreferred) 5′ antisense overhang; N indicatessubunits; [N] indicates that additional subunit pairs may be present;and P_(x), indicates a paring of sense N_(x) and antisense N_(x).Overhangs are not shown in the P diagram. In some embodiments a 3′ ASoverhang corresponds to region Z, the duplex region corresponds toregion X, and the 3′ S strand overhang corresponds to region Y, asdescribed elsewhere herein. (The diagram is not meant to imply maximumor minimum lengths, on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form aduplex are used at 1 or more of the positions in the duplex at the 5′end of the AS strand. The terminal pair (the most 5′ pair in terms ofthe AS strand) is designated as P⁻¹, and the subsequent pairingpositions (going in the 3′ direction in terms of the AS strand) in theduplex are designated, P⁻², P⁻³, P⁻⁴, P⁻⁵, and so on. The preferredregion in which to modify or modulate duplex formation is at P⁻⁵ throughP⁻¹, more preferably P⁻⁴ through P⁻¹, more preferably P⁻³ through P⁻¹Modification at P⁻¹, is particularly preferred, alone or withmodification(s) other position(s), e.g., any of the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions be chosenindependently from the group of:

-   -   A:U    -   G:U    -   I:C    -   mismatched pairs, e.g., non-canonical or other than canonical        pairings or pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve apairing which promotes dissociation will be made in the sense strand,though in some embodiments the change will be made in the antisensestrand.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote dissociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are G:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are mismatched pairs, e.g., non-canonical or other thancanonical pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotestability and inhibit dissociation or melting (e.g., on the free energyof association or dissociation of a particular pairing, the simplestapproach is to examine the pairs on an individual pair basis, thoughnext neighbor or similar analysis can also be used). In terms ofpromoting duplex stability:

G:C is preferred over A:U

-   -   Watson-Crick matches (A:T, A:U, G:C) are preferred over        non-canonical or other than canonical pairings    -   analogs that increase stability are preferred over Watson-Crick        matches (A:T, A:U, G:C)

2-amino-A:U is preferred over A:U 2-thio U or 5 are preferred over U:AMe-thio-U:A G-clamp (an analog of C having is preferred over C:G 4hydrogen bonds):G guanadinium-G-clamp:G is preferred over C:G pseudouridine:A is preferred over U:A

-   -   sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or        LNA, which enhance binding are preferred over non-modified        moieties and can be present on one or both strands to enhance        stability of the duplex. It is preferred that pairings which        increase the propensity to form a duplex are used at 1 or more        of the positions in the duplex at the 3′ end of the AS strand.        The terminal pair (the most 3′ pair in terms of the AS strand)        is designated as P₁, and the subsequent pairing positions (going        in the 5′ direction in terms of the AS strand) in the duplex are        designated, P₂, P₃, P₄, P₅, and so on. The preferred region in        which to modify to modulate duplex formation is at P₅ through        P₁, more preferably P₄ through P₁, more preferably P₃ through        P₁. Modification at P₁, is particularly preferred, alone or with        modification(s) at other position(s), e.g., any of the positions        just identified. It is preferred that at least 1, and more        preferably 2, 3, 4, or 5 of the pairs of the recited regions be        chosen independently from the group of:    -   G:C    -   a pair having an analog that increases stability over        Watson-Crick matches (A:T, A:U, G:C)    -   2-amino-A:U        2-thio U or 5 Me-thio-U:A    -   G-clamp (an analog of C having 4 hydrogen bonds):G    -   guanadinium-G-clamp:G    -   pseudo uridine:A    -   a pair in which one or both subunits has a sugar modification,        e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance        binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair having an analog that increases stability overWatson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-amino-A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are guanidinium-G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are pseudo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair in which one or both subunits has a sugarmodification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, whichenhances binding.

G-clamps and guanidinium G-clamps are discussed in the followingreferences: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-ClampContaining Oligonucleotides and Their Inhibition of the HIV-1 Tat-TARInteraction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262,2003; Holmes et al., “Steric inhibition of human immunodeficiency virustype-1 Tat-dependent trans-activation in vitro and in cells byoligonucleotides containing 2′-O-methyl G-clamp ribonucleosideanalogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al.,“Structural basis for recognition of guanosine by a synthetic tricycliccytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta,86:966-978, 2003; Rajeev, et al., “High-Affinity Peptide Nucleic AcidOligomers Containing Tricyclic Cytosine Analogues,” Organic Letters,4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- andGuanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002;Maier et al., “Nuclease resistance of oligonucleotides containing thetricyclic cytosine analogues phenoxazine and9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins of their nucleaseresistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al.,“A cytosine analog that confers enhanced potency to antisenseoligonucleotides,” Proceedings Of The National Academy Of Sciences OfThe United States Of America, 96:3513-8, 1999.

Simultaneously Decreasing the Stability of the AS 5′End of the Duplexand Increasing the Stability of the AS 3′ End of the Duplex

As is discussed above, an iRNA agent can be modified to both decreasethe stability of the AS 5′end of the duplex and increase the stabilityof the AS 3′ end of the duplex. This can be effected by combining one ormore of the stability decreasing modifications in the AS 5′ end of theduplex with one or more of the stability increasing modifications in theAS 3′ end of the duplex. Accordingly a preferred embodiment includesmodification in P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹ andmore preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularlypreferred, alone or with other position, e.g., the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions of the AS 5′ end ofthe duplex region be chosen independently from the group of:

-   -   A:U    -   G:U    -   I:C    -   mismatched pairs, e.g., non-canonical or other than canonical        pairings which include a universal base; and

a modification in P₅ through P₁, more preferably P₄ through P₁ and morepreferably P₃ through P₁. Modification at P₁, is particularly preferred,alone or with other position, e.g., the positions just identified. It ispreferred that at least 1, and more preferably 2, 3, 4, or 5 of thepairs of one of the recited regions of the AS 3′ end of the duplexregion be chosen independently from the group of:

-   -   G:C    -   a pair having an analog that increases stability over        Watson-Crick matches (A:T, A:U, G:C)    -   2-amino-A:U        2-thio U or 5 Me-thio-U:A    -   G-clamp (an analog of C having 4 hydrogen bonds):G    -   guanadinium-G-clamp:G    -   pseudo uridine:A    -   a pair in which one or both subunits has a sugar modification,        e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance        binding.

The invention also includes methods of selecting and making iRNA agentshaving DMTDS. E.g., when screening a target sequence for candidatesequences for use as iRNA agents one can select sequences having a DMTDSproperty described herein or one which can be modified, preferably withas few changes as possible, especially to the

AS strand, to provide a desired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence,and modifying at least one P in P⁻⁵ through P⁻¹ and/or at least one P inP₅ through P₁ to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., tosilence an SNCA RNA, to treat any disorder described herein, e.g., aneurodegenerative disorder, in any formulation described herein, andgenerally in and/or with the methods and compositions describedelsewhere herein. DMTDS iRNA agents can incorporate other modificationsdescribed herein, e.g., the attachment of targeting agents or theinclusion of modifications which enhance stability, e.g., the inclusionof nuclease resistant monomers or the inclusion of single strandoverhangs (e.g., 3′ AS overhangs and/or 3′ S strand overhangs) whichself associate to form intrastrand duplex structure.

Preferably these iRNA agents will have an architecture described herein.

OTHER EMBODIMENTS

An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g.,from exogenous DNA templates that are delivered into the cell. Forexample, the DNA templates can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc.Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of an iRNA agent and one that produces atranscript that includes the bottom strand of an iRNA agent. When thetemplates are transcribed, the iRNA agent is produced, and processedinto sRNA agent fragments that mediate gene silencing.

In vivo Delivery

An iRNA agent can be linked, e.g., noncovalently linked to a polymer forthe efficient delivery of the iRNA agent to a subject, e.g., a mammal,such as a human. The iRNA agent can, for example, be complexed withcyclodextrin. Cyclodextrins have been used as delivery vehicles oftherapeutic compounds. Cyclodextrins can form inclusion complexes withdrugs that are able to fit into the hydrophobic cavity of thecyclodextrin. In other examples, cyclodextrins form non-covalentassociations with other biologically active molecules such asoligonucleotides and derivatives thereof. The use of cyclodextrinscreates a water-soluble drug delivery complex, that can be modified withtargeting or other functional groups. Cyclodextrin cellular deliverysystem for oligonucleotides described in U.S. Pat. No. 5,691,316, whichis hereby incorporated by reference, are suitable for use in methods ofthe invention. In this system, an oligonucleotide is noncovalentlycomplexed with a cyclodextrin, or the oligonucleotide is covalentlybound to adamantine which in turn is non-covalently associated with acyclodextrin.

The delivery molecule can include a linear cyclodextrin copolymer or alinear oxidized cyclodextrin copolymer having at least one ligand boundto the cyclodextrin copolymer. Delivery systems, as described in U.S.Pat. No. 6,509,323, herein incorporated by reference, are suitable foruse in methods of the invention. An iRNA agent can be bound to thelinear cyclodextrin copolymer and/or a linear oxidized cyclodextrincopolymer. Either or both of the cyclodextrin or oxidized cyclodextrincopolymers can be crosslinked to another polymer and/or bound to aligand.

A composition for iRNA delivery can employ an “inclusion complex,” amolecular compound having the characteristic structure of an adduct. Inthis structure, the “host molecule” spatially encloses at least part ofanother compound in the delivery vehicle. The enclosed compound (the“guest molecule”) is situated in the cavity of the host molecule withoutaffecting the framework structure of the host. A “host” is preferablycyclodextrin, but can be any of the molecules suggested in U.S. PatentPubl. 2003/0008818, herein incorporated by reference.

Cyclodextrins can interact with a variety of ionic and molecularspecies, and the resulting inclusion compounds belong to the class of“host-guest” complexes. Within the host-guest relationship, the bindingsites of the host and guest molecules should be complementary in thestereoelectronic sense. A composition of the invention can contain atleast one polymer and at least one therapeutic agent, generally in theform of a particulate composite of the polymer and therapeutic agent,e.g., the iRNA agent. The iRNA agent can contain one or more complexingagents. At least one polymer of the particulate composite can interactwith the complexing agent in a host-guest or a guest-host interaction toform an inclusion complex between the polymer and the complexing agent.The polymer and, more particularly, the complexing agent can be used tointroduce functionality into the composition. For example, at least onepolymer of the particulate composite has host functionality and forms aninclusion complex with a complexing agent having guest functionality.Alternatively, at least one polymer of the particulate composite hasguest functionality and forms an inclusion complex with a complexingagent having host functionality. A polymer of the particulate compositecan also contain both host and guest functionalities and form inclusioncomplexes with guest complexing agents and host complexing agents. Apolymer with functionality can, for example, facilitate cell targetingand/or cell contact (e.g., targeting or contact to a neural cell),intercellular trafficking, and/or cell entry and release.

Upon forming the particulate composite, the iRNA agent may or may notretain its biological or therapeutic activity. Upon release from thetherapeutic composition, specifically, from the polymer of theparticulate composite, the activity of the iRNA agent is restored.Accordingly, the particulate composite advantageously affords the iRNAagent protection against loss of activity due to, for example,degradation and offers enhanced bioavailability. Thus, a composition maybe used to provide stability, particularly storage or solutionstability, to an iRNA agent or any active chemical compound. The iRNAagent may be further modified with a ligand prior to or afterparticulate composite or therapeutic composition formation. The ligandcan provide further functionality. For example, the ligand can be atargeting moiety.

Physiological Effects

The iRNA agents described herein can be designed such that determiningtherapeutic toxicity is made easier by the complementarity of the iRNAagent with both a human and a non-human animal sequence. By thesemethods, an iRNA agent can consist of a sequence that is fullycomplementary to a nucleic acid sequence from a human and a nucleic acidsequence from at least one non-human animal, e.g., a non-human mammal,such as a rodent, ruminant or primate. For example, the non-human mammalcan be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus,Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence ofthe iRNA agent could be complementary to sequences within homologousgenes, e.g., oncogenes or tumor suppressor genes, of the non-humanmammal and the human. By determining the toxicity of the iRNA agent inthe non-human mammal, one can extrapolate the toxicity of the iRNA agentin a human. For a more strenuous toxicity test, the iRNA agent can becomplementary to a human and more than one, e.g., two or three or more,non-human animals.

The methods described herein can be used to correlate any physiologicaleffect of an iRNA agent on a human, e.g., any unwanted effect, such as atoxic effect, or any positive, or desired effect.

Delivery Module

An RNA, e.g., an iRNA agent described herein, can be used with a drugdelivery conjugate or module, such as those described herein. Inaddition, an iRNA agent described herein, e.g., a palindromic iRNAagent, an iRNA agent having a non canonical pairing, an iRNA agent whichtargets a gene described herein, e.g., an SNCA gene, an iRNA agenthaving a chemical modification described herein, e.g., a modificationwhich enhances resistance to degradation, an iRNA agent having anarchitecture or structure described herein, an iRNA agent administeredas described herein, or an iRNA agent formulated as described herein,combined with, associated with, and delivered by such a drug deliveryconjugate or module.

The iRNA agents can be complexed to a delivery agent that features amodular complex. The complex can include a carrier agent linked to oneor more of (preferably two or more, more preferably all three of): (a) acondensing agent (e.g., an agent capable of attracting, e.g., binding, anucleic acid, e.g., through ionic or electrostatic interactions); (b) afusogenic agent (e.g., an agent capable of fusing and/or beingtransported through a cell membrane, e.g., an endosome membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type such as a neural cell in the brain.

An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can belinked, e.g., coupled or bound, to the modular complex. The iRNA agentcan interact with the condensing agent of the complex, and the complexcan be used to deliver an iRNA agent to a cell, e.g., in vitro or invivo. For example, the complex can be used to deliver an iRNA agent to asubject in need thereof, e.g., to deliver an iRNA agent to a subjecthaving a disorder, e.g., a disorder described herein, such as aneurodegenerative disease or disorder.

The fusogenic agent and the condensing agent can be different agents orthe one and the same agent. For example, a polyamino chain, e.g.,polyethyleneimine (PEI), can be the fusogenic and/or the condensingagent.

The delivery agent can be a modular complex. For example, the complexcan include a carrier agent linked to one or more of (preferably two ormore, more preferably all three of):

(a) a condensing agent (e.g., an agent capable of attracting, e.g.,binding, a nucleic acid, e.g., through ionic interaction),

(b) a fusogenic agent (e.g., an agent capable of fusing and/or beingtransported through a cell membrane, e.g., an endosome membrane), and

(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type such as a neural cell (e.g., a neural cell in thebrain). A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyamino acids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGDpeptide or RGD peptide mimetic.

Carrier agents. The carrier agent of a modular complex described hereincan be a substrate for attachment of one or more of: a condensing agent,a fusogenic agent, and a targeting group. The carrier agent wouldpreferably lack an endogenous enzymatic activity. The agent wouldpreferably be a biological molecule, preferably a macromolecule.Polymeric biological carriers are preferred. It would also be preferredthat the carrier molecule be biodegradable.

The carrier agent can be a naturally occurring substance, such as aprotein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL),or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,insulin, cyclodextrin or hyaluronic acid); or lipid. The carriermolecule can also be a recombinant or synthetic molecule, such as asynthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Other useful carrier molecules can be identified byroutine methods.

A carrier agent can be characterized by one or more of: (a) is at least1 Da in size; (b) has at least 5 charged groups, preferably between 5and 5000 charged groups; (c) is present in the complex at a ratio of atleast 1:1 carrier agent to fusogenic agent; (d) is present in thecomplex at a ratio of at least 1:1 carrier agent to condensing agent;(e) is present in the complex at a ratio of at least 1:1 carrier agentto targeting agent.

Fusogenic agents. A fusogenic agent of a modular complex describedherein can be an agent that is responsive to, e.g., changes chargedepending on, the pH environment. Upon encountering the pH of anendosome, it can cause a physical change, e.g., a change in osmoticproperties which disrupts or increases the permeability of the endosomemembrane. Preferably, the fusogenic agent changes charge, e.g., becomesprotonated, at pH lower than physiological range. For example, thefusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agentcan serve to release the iRNA agent into the cytoplasm of a cell afterthe complex is taken up, e.g., via endocytosis, by the cell, therebyincreasing the cellular concentration of the iRNA agent in the cell.

In one embodiment, the fusogenic agent can have a moiety, e.g., an aminogroup, which, when exposed to a specified pH range, will undergo achange, e.g., in charge, e.g., protonation. The change in charge of thefusogenic agent can trigger a change, e.g., an osmotic change, in avesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, thefusogenic agent, upon being exposed to the pH environment of anendosome, will cause a solubility or osmotic change substantial enoughto increase the porosity of (preferably, to rupture) the endosomalmembrane.

The fusogenic agent can be a polymer, preferably a polyamino chain,e.g., polyethyleneimine (PEI). The PEI can be linear, branched,synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, orlipid substituted PEI.

In other embodiments, the fusogenic agent can be polyhistidine,polyimidazole, polypyridine, polypropyleneimine, mellitin, or apolyacetal substance, e.g., a cationic polyacetal. In some embodiment,the fusogenic agent can have an alpha helical structure. The fusogenicagent can be a membrane disruptive agent, e.g., mellittin.

A fusogenic agent can have one or more of the following characteristics:(a) is at least 1 Da in size; (b) has at least 10 charged groups,preferably between 10 and 5000 charged groups, more preferably between50 and 1000 charged groups; (c) is present in the complex at a ratio ofat least 1:1 fusogenic agent to carrier agent; (d) is present in thecomplex at a ratio of at least 1:1 fusogenic agent to condensing agent;(e) is present in the complex at a ratio of at least 1:1 fusogenic agentto targeting agent.

Other suitable fusogenic agents can be tested and identified by askilled artisan. The ability of a compound to respond to, e.g., changecharge depending on, the pH environment can be tested by routinemethods, e.g., in a cellular assay. For example, a test compound iscombined or contacted with a cell, and the cell is allowed to take upthe test compound, e.g., by endocytosis. An endosome preparation canthen be made from the contacted cells and the endosome preparationcompared to an endosome preparation from control cells. A change, e.g.,a decrease, in the endosome fraction from the contacted cell vs. thecontrol cell indicates that the test compound can function as afusogenic agent. Alternatively, the contacted cell and control cell canbe evaluated, e.g., by microscopy, e.g., by light or electronmicroscopy, to determine a difference in endosome population in thecells. The test compound can be labeled. In another type of assay, amodular complex described herein is constructed using one or more testor putative fusogenic agents. The modular complex can be constructedusing a labeled nucleic acid instead of the iRNA. A two-step assay canbe performed, wherein a first assay evaluates the ability of a testcompound alone to respond to, e.g., change charge depending on, the pHenvironment; and a second assay evaluates the ability of a modularcomplex that includes the test compound to respond to, e.g., changecharge depending on, the pH environment.

Condensing agent. The condensing agent of a modular complex describedherein can interact with (e.g., attracts, holds, or binds to) an iRNAagent and act to (a) condense, e.g., reduce the size or charge of theiRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNAagent against degradation. The condensing agent can include a moiety,e.g., a charged moiety, that can interact with a nucleic acid, e.g., aniRNA agent, e.g., by ionic interactions. The condensing agent wouldpreferably be a charged polymer, e.g., a polycationic chain. Thecondensing agent can be a polylysine (PLL), spermine, spermidine,polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimerpolyamine, arginine, amidine, protamine, cationic lipid, cationicporphyrin, quarternary salt of a polyamine, or an alpha helical peptide.

A condensing agent can have the following characteristics: (a) at least1 Da in size; (b) has at least 2 charged groups, preferably between 2and 100 charged groups; (c) is present in the complex at a ratio of atleast 1:1 condensing agent to carrier agent; (d) is present in thecomplex at a ratio of at least 1:1 condensing agent to fusogenic agent;(e) is present in the complex at a ratio of at least 1:1 condensingagent to targeting agent.

Other suitable condensing agents can be tested and identified by askilled artisan, e.g., by evaluating the ability of a test agent tointeract with a nucleic acid, e.g., an iRNA agent. The ability of a testagent to interact with a nucleic acid, e.g., an iRNA agent, e.g., tocondense or protect the iRNA agent, can be evaluated by routinetechniques. In one assay, a test agent is contacted with a nucleic acid,and the size and/or charge of the contacted nucleic acid is evaluated bya technique suitable to detect changes in molecular mass and/or charge.Such techniques include non-denaturing gel electrophoresis,immunological methods, e.g., immunoprecipitation, gel filtration, ionicinteraction chromatography, and the like. A test agent is identified asa condensing agent if it changes the mass and/or charge (preferablyboth) of the contacted nucleic acid, compared to a control. A two-stepassay can also be performed, wherein a first assay evaluates the abilityof a test compound alone to interact with, e.g., bind to, e.g., condensethe charge and/or mass of, a nucleic acid; and a second assay evaluatesthe ability of a modular complex that includes the test compound tointeract with, e.g., bind to, e.g., condense the charge and/or mass of,a nucleic acid.

Amphipathic Delivery Agents

An RNA, e.g., an iRNA agent, described herein can be used with anamphipathic delivery conjugate or module, such as those describedherein. In addition, an iRNA agent described herein, e.g., a palindromiciRNA agent, an iRNA agent having a noncanonical pairing, an iRNA agentwhich targets a gene described herein, e.g., an SNCA gene, an iRNA agenthaving a chemical modification described herein, e.g., a modificationwhich enhances resistance to degradation, an iRNA agent having anarchitecture or structure described herein, an iRNA agent administeredas described herein, or an iRNA agent formulated as described herein,combined with, associated with, and delivered by such an amphipathicdelivery conjugate.

An amphipathic molecule is a molecule having a hydrophobic and ahydrophilic region. Such molecules can interact with (e.g., penetrate ordisrupt) lipids, e.g., a lipid bilayer of a cell. As such, they canserve as delivery agent for an associated (e.g., bound) iRNA (e.g., aniRNA or sRNA described herein). A preferred amphipathic molecule to beused in the compositions described herein (e.g., the amphipathic iRNAconstructs described herein) is a polymer. The polymer may have asecondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide,e.g., a polypeptide having a secondary structure such that thepolypeptide has a hydrophilic and a hybrophobic face. The design ofamphipathic peptide structures (e.g., alpha-helical polypeptides) isroutine to one of skill in the art. For example, the followingreferences provide guidance: Grell et al. (2001) J Pept Sci 7(3):146-51;Chen et al. (2002) J Pept Res 59(1):18-33; Iwata et al. (1994) J BiolChem 269(7):4928-33; Cornut et al. (1994) FEBS Lett 349(1):29-33;Negrete et al. (1998) Protein Sci 7(6):1368-79.

Another example of an amphipathic polymer is a polymer made up of two ormore amphipathic subunits, e.g., two or more subunits containing cyclicmoieties (e.g., a cyclic moiety having one or more hydrophilic groupsand one or more hydrophobic groups). For example, the subunit maycontain a steroid, e.g., cholic acid; or a aromatic moiety. Suchmoieties preferably can exhibit atropisomerism, such that they can formopposing hydrophobic and hydrophilic faces when in a polymer structure.

The ability of a putative amphipathic molecule to interact with a lipidmembrane, e.g., a cell membrane, can be tested by routine methods, e.g.,in a cell free or cellular assay. For example, a test compound iscombined or contacted with a synthetic lipid bilayer, a cellularmembrane fraction, or a cell, and the test compound is evaluated for itsability to interact with, penetrate, or disrupt the lipid bilayer, cellmembrane or cell. The test compound can be labeled in order to detectthe interaction with the lipid bilayer, cell membrane, or cell. Inanother type of assay, the test compound is linked to a reportermolecule or an iRNA agent (e.g., an iRNA or sRNA described herein), andthe ability of the reporter molecule or iRNA agent to penetrate thelipid bilayer, cell membrane or cell is evaluated. A two-step assay canalso be performed, wherein a first assay evaluates the ability of a testcompound alone to interact with a lipid bilayer, cell membrane or cell;and a second assay evaluates the ability of a construct (e.g., aconstruct described herein) that includes the test compound and areporter or iRNA agent to interact with a lipid bilayer, cell membraneor cell.

An amphipathic polymer useful in the compositions described herein hasat least 2, preferably at least 5, more preferably at least 10, 25, 50,100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids orcyclic subunits). A single amphipathic polymer can be linked to one ormore, e.g., 2, 3, 5, 1 0 or more iRNA agents (e.g., iRNA or sRNA agentsdescribed herein). In some embodiments, an amphipathic polymer cancontain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNAagent (e.g., an iRNA or sRNA agent described herein) linked to anamphipathic molecule. The iRNA agent and the amphipathic molecule may beheld in continuous contact with one another by either covalent ornoncovalent linkages.

The amphipathic molecule of the composition or construct is preferablyother than a phospholipid, e.g., other than a micelle, membrane ormembrane fragment.

The amphipathic molecule of the composition or construct is preferably apolymer. The polymer may include two or more amphipathic subunits. Oneor more hydrophilic groups and one or more hydrophobic groups may bepresent on the polymer. The polymer may have a repeating secondarystructure as well as a first face and a second face. The distribution ofthe hydrophilic groups and the hydrophobic groups along the repeatingsecondary structure can be such that one face of the polymer is ahydrophilic face and the other face of the polymer is a hydrophobicface.

The amphipathic molecule can be a polypeptide, e.g., a polypeptidecomprising an a-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunitscontaining one or more cyclic moiety (e.g., a cyclic moiety having oneor more hydrophilic groups and/or one or more hydrophobic groups). Inone embodiment, the polymer is a polymer of cyclic moieties such thatthe moieties have alternating hydrophobic and hydrophilic groups. Forexample, the subunit may contain a steroid, e.g., cholic acid. Inanother example, the subunit may contain an aromatic moiety. Thearomatic moiety may be one that can exhibit atropisomerism, e.g., a2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

Referring to Formula M, R₁ is C₁-C₁₀₀ alkyl optionally substituted witharyl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted withO, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonicacid; OR₆; or C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo,nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonicacid, phosphate, phosphate ester, substituted or unsubstituted aryl,carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/oroptionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

R₃ is hydrogen, or when taken together with R₄ forms a fused phenylring.

R₄ is hydrogen, or when taken together with R₃ forms a fused phenylring.

R₅ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl,alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl;or C₁-C₁₀₀ perfluoroalkyl; and R₆ is C₁-C₁₀₀ alkyl optionallysubstituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl oralkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester,substituted or unsubstituted aryl, carboxyl, carboxylate, aminocarbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S,S(O), SO₂, alkenyl, or alkynyl.

Increasing Cellular Uptake of dsRNAs

A method of the invention that can include the administration of an iRNAagent and a drug that affects the uptake of the iRNA agent into thecell. The drug can be administered before, after, or at the same timethat the iRNA agent is administered. The drug can be covalently linkedto the iRNA agent. The drug can have a transient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, forexample, by disrupting the cell's cytoskeleton, e.g., by disrupting thecell's microtubules, microfilaments, and/or intermediate filaments. Thedrug can be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a secondagent. For example, an iRNA agent used to treat a particular disordercan be coupled to a second therapeutic agent, e.g., an agent other thanthe iRNA agent. The second therapeutic agent can be one which isdirected to the treatment of the same disorder. For example, in the caseof an iRNA used to treat a disorder characterized by alpha-synucleinaggregates, e.g., PD, the iRNA agent can be coupled to a second agentwhich is useful for the treatment of PD.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods.Exemplary methods include: organic synthesis and RNA cleavage, e.g., invitro cleavage.

Organic Synthesis. An iRNA can be made by separately synthesizing eachrespective strand of a double-stranded RNA molecule. The componentstrands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB(Uppsala Sweden), can be used to produce a large amount of a particularRNA strand for a given iRNA. The OligoPilotII reactor can efficientlycouple a nucleotide using only a 1.5 molar excess of a phosphoramiditenucleotide. To make an RNA strand, ribonucleotides amidites are used.Standard cycles of monomer addition can be used to synthesize the 21 to23 nucleotide strand for the iRNA. Typically, the two complementarystrands are produced separately and then annealed, e.g., after releasefrom the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. Thecomplementary of the species to a particular target gene can beprecisely specified. For example, the species may be complementary to aregion that includes a polymorphism, e.g., a single nucleotidepolymorphism. Further the location of the polymorphism can be preciselydefined. In some embodiments, the polymorphism is located in an internalregion, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of thetermini.

dsRNA Cleavage. iRNAs can also be made by cleaving a larger ds iRNA. Thecleavage can be mediated in vitro or in vivo. For example, to produceiRNAs by cleavage in vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid(DNA) segment in both directions. For example, the HiScribe™ RNAitranscription kit (New England Biolabs) provides a vector and a methodfor producing a dsRNA for a nucleic acid segment that is cloned into thevector at a position flanked on either side by a T7 promoter. Separatetemplates are generated for T7 transcription of the two complementarystrands for the dsRNA. The templates are transcribed in vitro byaddition of T7 RNA polymerase and dsRNA is produced. Similar methodsusing PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) canalso be used. In one embodiment, RNA generated by this method iscarefully purified to remove endotoxins that may contaminatepreparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example,using a Dicer or comparable RNAse III-based activity. For example, thedsRNA can be incubated in an in vitro extract from Drosophila or usingpurified components, e.g. a purified RNAse or RISC complex (RNA-inducedsilencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15;15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, eachbeing a particular 21 to 23 nt fragment of a source dsRNA molecule. Forexample, iRNAs that include sequences complementary to overlappingregions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussedbelow.

Formulation

The iRNA agents described herein can be formulated for administration toa subject.

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration.

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA that can mediated RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least three, five, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA). Forexample, a iRNA composition for the treatment of a neurodegenerativedisease, e.g. PD, might include a known PD therapeutic (e.g., levadopaor depronil)

Targeting

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAs. Itshould be understood, however, that these formulations, compositions andmethods can be practiced with other iRNA agents, e.g., modified iRNAagents, and such practice is within the invention.

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent,or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into a sRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)is targeted to a particular cell. For example, a liposome or particle orother structure that includes a iRNA can also include a targeting moietythat recognizes a specific molecule on a target cell. The targetingmoiety can be a molecule with a specific affinity for a target cell.Targeting moieties can include antibodies directed against a proteinfound on the surface of a target cell, or the ligand or areceptor-binding portion of a ligand for a molecule found on the surfaceof a target cell.

An antigen, can be used to target an iRNA to a neural cell in the brain.

In one embodiment, the targeting moiety is attached to a liposome. Forexample, U.S. Pat. No. 6,245,427 describes a method for targeting aliposome using a protein or peptide. In another example, a cationiclipid component of the liposome is derivatized with a targeting moiety.For example, WO 96/37194 describes convertingN-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimideactivated ester. The product was then coupled to an RGD peptide.

Antibodies

An composition described herein can include an antibody that targets asynuclein polypeptide, e.g., to block synuclein activity and/or inhibitsynuclein aggregation. An antibody can be an antibody or a fragmentthereof, e.g., an antigen binding portion thereof. As used herein, theterm “antibody” refers to a protein comprising at least one, andpreferably two, heavy (H) chain variable regions (abbreviated herein asVH), and at least one and preferably two light (L) chain variableregions (abbreviated herein as VL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). The extent of the frameworkregion and CDR's has been precisely defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242,1991, and Chothia et al., J. Mol. Biol. 196:901-917, 1987, which areincorporated herein by reference). Each VH and VL is composed of threeCDR's and four FRs, arranged from amino-terminus to carboxyl-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The antibody can further include a heavy and light chain constantregion, to thereby form a heavy and light immunoglobulin chain,respectively. In one embodiment, the antibody is a tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains, wherein theheavy and light immunoglobulin chains are inter-connected by, e.g.,disulfide bonds. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. The light chain constant region is comprisedof one domain, CL. The variable region of the heavy and light chainscontains a binding domain that interacts with an antigen. The constantregions of the antibodies typically mediate the binding of the antibodyto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (Clq) of the classicalcomplement system.

The term “antigen-binding fragment” of an antibody (or simply “antibodyportion,” or “fragment”), as used herein, refers to one or morefragments of a full-length antibody that retain the ability tospecifically bind to an antigen (e.g., a polypeptide encoded by an SNCAnucleic acid). Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., Nature 341:544-546, 1989), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate nucleic acids, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883, 1988). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding fragment” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular protein with whichit immunoreacts.

Anti-protein/anti-peptide antisera or monoclonal antibodies can be madeas described herein by using standard protocols (See, for example,Antibodies: A Laboratory Manual ed. by Harlow and Lane, Cold SpringHarbor Press, 1988).

A protein described herein, e.g., an alpha synuclein polypeptide, can beused as an immunogen to generate antibodies that bind the componentusing standard techniques for polyclonal and monoclonal antibodypreparation. The full-length component protein can be used or,alternatively, antigenic peptide fragments of the component can be usedas immunogens.

Typically, a peptide is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, a recombinant form of a protein described herein, e.g., analpha-synuclein polypeptide. See, e.g., U.S. Pat. No. 5,460,959; andco-pending U.S. applications U.S. Ser. No. 08/334,797; U.S. Ser. No.08/231,439; U.S. Ser. No. 08/334,455; and U.S. Ser. No. 08/928,881,which are hereby expressly incorporated by, reference in their entirety.The nucleotide and amino acid sequences of alpha-synuclein are known.The preparation can further include an adjuvant, such as Freund'scomplete or incomplete adjuvant, or similar immunostimulatory agent.Immunization of a suitable subject with an immunogenic protein describedherein, e.g., an alpha-synuclein polypeptide, or fragment preparationinduces a polyclonal antibody response.

Additionally, antibodies produced by genetic engineering methods, suchas chimeric and humanized monoclonal antibodies, comprising both humanand non-human portions, which can be made using standard recombinant DNAtechniques, can be used. Such chimeric and humanized monoclonalantibodies can be produced by genetic engineering using standard DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al., Science 240:1041-1043, 1988; Liu etal., PNAS 84:3439-3443, 1987; Liu et al., J. Immunol. 139:3521-3526,1987; Sun et al., PNAS 84:214-218, 1987; Nishimura et al., Canc. Res.47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985; and Shaw etal., J. Natl. Cancer Inst. 80:1553-1559, 1988); Morrison, S. L., Science229:1202-1207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter U.S.Pat. No. 5,225,539; Jones et al., Nature 321:552-525, 1986; Verhoeyan etal., Science 239:1534, 1988; and Beidler et al., J. Immunol.141:4053-4060, 1988.

In addition, a human monoclonal antibody directed against a proteindescribed herein, e.g., an alpha-synuclein protein, can be made usingstandard techniques. For example, human monoclonal antibodies can begenerated in transgenic mice or in immune deficient mice engrafted withantibody-producing human cells. Methods of generating such mice aredescribe, for example, in Wood et al. PCT publication WO 91/00906;Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. PCTpublication WO 92/03918; Kay et al. PCT publication WO 92/03917; Kay etal. PCT publication WO 93/12227; Kay et al. PCT publication WO 94/25585;Rajewsky et al. PCT publication WO 94/04667; Ditullio et al. PCTpublication WO 95/17085; Lonberg et al., Nature 368:856-859, 1994; Greenet al., Nature Genet. 7:13-21, 1994; Morrison et al., Proc. Natl. Acad.Sci. USA 81:6851-6855, 1994; Bruggeman et al., Year Immunol 7:33-40,1993; Choi et al., Nature Genet. 4:117-123, 1993; Tuaillon et al., PNAS90:3720-3724, 1993; Bruggeman et al., Eur J Immunol 21:1323-1326, 1991;Duchosal et al. PCT publication WO 93/05796; U.S. Pat. No. 5,411,749;McCune et al., Science 241:1632-1639, 1988; Kamel-Reid et al., Science242:1706, 1988; Spanopoulou, Genes & Development 8:1030-1042, 1994; andShinkai et al., Cell 68:855-868, 1992. A human antibody-transgenic mouseor an immune deficient mouse engrafted with human antibody-producingcells or tissue can be immunized with a protein described herein, e.g.,an alpha-synuclein protein, or an antigenic peptide thereof, andsplenocytes from these immunized mice can then be used to createhybridomas. Methods of hybridoma production are well known.

Human monoclonal antibodies against a protein described herein, e.g., analpha-synuclein polypeptide, can also be prepared by constructing acombinatorial immunoglobulin library, such as a Fab phage displaylibrary or an scFv phage display library, using immunoglobulin lightchain and heavy chain cDNAs prepared from mRNA derived from lymphocytesof a subject. See, e.g., McCafferty et al. PCT publication WO 92/01047;Marks et al., J. Mol. Biol. 222:581-597, 1991; and Griffiths et al.,EMBO J. 12:725-734, 1993. In addition, a combinatorial library ofantibody variable regions can be generated by mutating a known humanantibody. For example, a variable region of a human antibody known tobind a protein described herein can be mutated by, for example, usingrandomly altered mutagenized oligonucleotides, to generate a library ofmutated variable regions which can then be screened to bind to a proteindescribed herein, e.g., an alpha-synuclein. Methods of inducing randommutagenesis within the CDR regions of immunoglobulin heavy and/or lightchains, methods of crossing randomized heavy and light chains to formpairings and screening methods can be found in, for example, Barbas etal. PCT publication WO 96/07754; and Barbas et al., Proc. Nat'l Acad.Sci. USA 89:4457-4461, 1992.

The immunoglobulin library can be expressed by a population of displaypackages, preferably derived from filamentous phage, to form an antibodydisplay library. Examples of methods and reagents particularly amenablefor use in generating an antibody display library can be found in, forexample, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTpublication WO 92/18619; Dower et al. PCT publication WO 91/17271;Winter et al. PCT publication WO 92/20791; Markland et al. PCTpublication WO 92/15679; Breitling et al. PCT publication WO 93/01288;McCafferty et al. PCT publication WO 92/01047; Garrard et al. PCTpublication WO 92/09690; Ladner et al. PCT publication WO 90/02809;Fuchs et al., Bio/Technology 9:1370-1372; 1991; Hay et al., Hum AntibodHybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989;Griffiths et al., 1993, supra; Hawkins et al., J Mol Biol 226:889-896,1992; Clackson et al., Nature 352:624-628, 1991; Gram et al., PNAS89:3576-3580, 1992; Garrad et al., Bio/Technology 9:1373-1377, 1991;Hoogenboom et al., Nuc Acid Res 19:4133-4137, 1991; and Barbas et al.,PNAS 88:7978-7982, 1991. Once displayed on the surface of a displaypackage (e.g., filamentous phage), the antibody library is screened toidentify and isolate packages that express an antibody that binds aprotein described herein, e.g., an alpha-synuclein polypeptide. In apreferred embodiment, the primary screening of the library involvespanning with an immobilized protein described herein, and displaypackages expressing antibodies that bind immobilized proteins describedherein are selected.

Antisense Nucleic Acid Sequences

Nucleic acid molecules that are antisense to a nucleotide encoding aprotein described herein, e.g., an alpha-synuclein polypeptide, can alsobe used as an agent that inhibits expression of the protein. An“antisense” nucleic acid includes a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding the component, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can form hydrogen bonds with a sense nucleic acid. The antisensenucleic acid can be complementary to a portion of a coding strand or thenoncoding strand.

The coding strand sequences encoding alpha-synuclein proteins are known.Given a coding strand sequence (e.g., the sequence of a sense strand ofa cDNA molecule), antisense nucleic acids can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to a portion of the coding or noncodingregion of mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofthe mRNA, e.g., the 5′ UTR. An antisense oligonucleotide can be, forexample, about 10 to 25 nucleotides in length (e.g., 11, 12, 13, 14, 15,16, 18, 19, 20, or 24 nucleotides in length).

An antisense nucleic acid can be constructed using chemical synthesisand enzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides that can be used to generate theantisense nucleic acid include 2′-O-methylated nucleotides,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

An antisense agent can include ribonucleotides only,deoxyribonucleotides only (e.g., oligodeoxynucleotides), or bothdeoxyribonucleotide and ribonucleotide sequences. For example, anantisense agent consisting only of ribonucleotides can hybridize to acomplementary RNA, e.g., an alpha-synuclein RNA, and prevent access ofthe translation machinery to the target RNA transcript, therebypreventing protein synthesis. An antisense molecule including onlydeoxyribonucleotides, or deoxyribonucleotides and ribonucleotides, e.g.,DNA sequence flanked by RNA sequence at the 5′ and 3′ ends of theantisense agent, can hybridize to a complementary RNA, and the RNAtarget can be subsequently cleaved by an enzyme, e.g., RNAseH.Degradation of the target RNA prevents translation. The flanking RNAsequences can include 2′-O-methylated nucleotides, and phosphorothioatelinkages, and the internal DNA sequence can include phosphorothioateinternucleotide linkages. The internal DNA sequence is preferably atleast five nucleotides in length when targeting by RNAseH activity isdesired.

For increased nuclease resistance, an antisense agent can be furthermodified by inverting the nucleoside at the 3′-terminus with a 3′-3′linkage. In another alternative, the 3′-terminus can be blocked with anaminoalkyl group.

Zinc Finger Proteins (ZFPs)

Zinc finger protein technology can be used to down-regulatetranscription of a candidate target gene, e.g., an SNCA gene. Forexample, an SNCA gene-specific DNA binding domain can be fused to arepressor domain to down-regulate SNCA gene expression. Zinc fingerproteins can be assembled using variable numbers of zinc finger domainsof varied specificity providing DNA binding proteins that not onlyrecognize novel sequences but also sequences of varied length. Zincfinger binding proteins for the regulation of gene expression aredescribed, for example, in U.S. Pat. Nos. 6,607,882, and 6,534,261.

The target site recognized by a ZFP can be any suitable site in thetarget gene (e.g., the SNCA gene) that will allow repression of geneexpression by a ZFP, optionally linked to a regulatory domain. Preferredtarget sites include regions adjacent to, downstream, or upstream of thetranscription start site. In addition, target sites can also be locatedin enhancer regions, repressor sites, RNA polymerase pause sites, andspecific regulatory sites (e.g., a REP1 site), sites in the cDNAencoding region or in an expressed sequence tag (EST) coding region.Typically each finger recognizes 2-4 base pairs, with a two finger ZFPbinding to a 4 to 7 bp target site, a three finger ZFP binding to a 6 to10 base pair site, and a six finger ZFP binding to two adjacent targetsites, each target site having from about 6-10 base pairs.

Typically, the zinc finger DNA-binding domain is linked to a regulatorydomain, e.g, a transcription factor repressor domain such as theKruppel-associated box (KRAB), the ERF repressor domain (ERD), or themSIN3 interaction domain (SID). For repression of gene expression,typically the expression of the gene is reduced by about 20% (i.e., 80%of non-ZFP modulated expression), more preferably by about 50% (i.e.,50% of non-ZFP modulated expression), more preferably by about 75-100%(i.e., 25% to 0% of non-ZFP modulated expression).

A zinc finger protein can be engineered to respond to a small molecule,such that the small molecule can regulate activity of the zinc fingerprotein. In one embodiment, a cell comprises two zinc finger proteins.The zinc finger proteins can target two different candidate genes. Forexample, one ZFP can target an SNCA gene to inhibit expression, and asecond ZFP can target a gene encoding a component of the proteosomemachinery, e.g, to enhance expression. Alternatively, the second ZFP cantarget and inhibit expression of a second gene that contributes to thealpha-synuclein aggregation phenotype. Alternatively, the zinc fingerproteins can target two different target sites on the same candidategene. Expression of each zinc finger protein can be under small moleculecontrol (e.g., by two different small molecules) to allow for variationsin the degree of repression of gene expression.

A “small molecule,” as used herein is a chemical compound that canaffect the phenotype of a cell or organism by, for example, modulatingthe activity of a specific protein or nucleic acid, e.g., an SNCAprotein or nucleic acid, within a cell. Small molecules may affect acell by directly interacting with a protein or by interacting with amolecule that acts upstream or downstream of the biochemical cascadethat results in protein expression or activity. Typically, a smallmolecule has a molecular weight of less than about 3000, preferably lessthan about 2000, more preferably less than about 1000, less than about900, less than about 800, less than about 700 or less than about 600 Da.

Treatment Methods and Routes of Delivery

The following discussion refers to treatment with an iRNA agent.However, it is to be understood that the invention includes analogousmethods and compositions which use or embody other inhibitory agentsdisclosed herein, e.g., antisense molecules and ribozymes that targetSNCA RNA, zinc finger proteins, and antibodies, and synthetic andnaturally-occurring polypeptides, or small molecules that, in preferredembodiments, bind to and inhibit the SNCA protein. A composition thatincludes a composition targeting alpha-synuclein, e.g., a ribozyme,antisense oligonucleotide, iRNA agent, antibody, small molecule, or zincfinger protein, can be delivered to a subject by a variety of routes.Exemplary routes include intrathecal, parenchymal (e.g., in the brain),intravenous, nasal, and ocular delivery. A preferred route of deliveryis directly to the brain. The anti-SNCA agents can be incorporated intopharmaceutical compositions suitable for administration. For example,compositions can include one or more species of an iRNA agent and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, intranasal,transdermal), oral or parenteral. Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, or intrathecal or intraventricular administration.

The route of delivery can be dependent on the disorder of the patient.For example, a subject diagnosed with PD can be administered ananti-SNCA iRNA agent directly to the brain, e.g., directly to thesubstantia nigra of the brain (e.g., into the striatal dopamine domainswithin the substantia nigra). A subject diagnosed with multiple systematrophy can be administered an iRNA agent directly into the brain, e.g.,into the striatum and substantia nigra regions of the brain, and intothe spinal cord. A subject diagnosed with Lewy body dementia can beadministered an iRNA agent directly into the brain, e.g., directly intothe cortex of the brain, and administration can be diffuse. In additionto an agent which inhibits SNCA expression, e.g., an anti-SNCA iRNAagent, a patient can be administered a second therapy, e.g., apalliative therapy and/or disease-specific therapy. A palliative therapycan be a dopaminergic therapy, for example, such as methyldopa orcoenzyme Q10.

In some embodiments, such as for the treatment of Parkinson's Disease,the secondary therapy can be, for example, symptomatic (e.g., foralleviating symptoms), neuroprotective (e.g., for slowing or haltingdisease progression), or restorative (e.g., for reversing the diseaseprocess). Symptomatic therapies include the drugs carbidopa/levodopa,entacapone, tolcapone, pramipexole, ropinerole, pergolide,bromocriptine, selegeline, amantadine, and several anticholingergicagents. Deep brain stimulation surgery as well as stereotactic brainlesioning may also provide symptomatic relief. Neuroprotective therapiesinclude, for example, carbidopa/levodopa, selegeline, vitamin E,amantadine, pramipexole, ropinerole, coenzyme Q10, and GDNF. Restorativetherapies can include, for example, surgical transplantation of stemcells.

An anti-SNCA iRNA agent can be delivered to neural cells of the brain.Delivery methods that do not require passage of the composition acrossthe blood-brain barrier can be utilized. For example, a pharmaceuticalcomposition containing an iRNA agent can be delivered to the patient byinjection directly into the area containing the alpha-synucleinaggregates. For example, the pharmaceutical composition can be deliveredby injection directly into the brain. The injection can be bystereotactic injection into a particular region of the brain (e.g., thesubstantia nigra, cortex, hippocampus, or globus pallidus). The iRNAagent can be delivered into multiple regions of the central nervoussystem (e.g., into multiple regions of the brain, and/or into the spinalcord). The iRNA agent can be delivered into diffuse regions of the brain(e.g., diffuse delivery to the cortex of the brain).

In one embodiment, the iRNA agent can be delivered by way of a cannulaor other delivery device having one end implanted in a tissue, e.g., thebrain, e.g., the substantia nigra, cortex, hippocampus, or globuspallidus of the brain. The cannula can be connected to a reservoir ofiRNA agent. The flow or delivery can be mediated by a pump, e.g., anosmotic pump or minipump. In one embodiment, a pump and reservoir areimplanted in an area distant from the tissue, e.g., in the abdomen, anddelivery is effected by a conduit leading from the pump or reservoir tothe site of release. Devices for delivery to the brain are described,for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014.

An iRNA agent can be modified such that it is capable of traversing theblood brain barrier. For example, the iRNA agent can be conjugated to amolecule that enables the agent to traverse the barrier. Such modifiediRNA agents can be administered by any desired method, such as byintraventricular or intramuscular injection, or by pulmonary delivery,for example.

The anti-SNCA iRNA agent can be administered ocularly, such as to treatretinal disorder, e.g., a retinopathy. For example, the pharmaceuticalcompositions can be applied to the surface of the eye or nearby tissue,e.g., the inside of the eyelid. They can be applied topically, e.g., byspraying, in drops, as an eyewash, or an ointment. Ointments ordroppable liquids may be delivered by ocular delivery systems known inthe art such as applicators or eye droppers. Such compositions caninclude mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers. The pharmaceutical compositioncan also be administered to the interior of the eye, and can beintroduced by a needle or other delivery device which can introduce itto a selected area or structure. The composition containing the iRNAagent can also be applied via an ocular patch.

Administration can be provided by the subject or by another person,e.g., a another caregiver. A caregiver can be any entity involved withproviding care to the human: for example, a hospital, hospice, doctor'soffice, outpatient clinic; a healthcare worker such as a doctor, nurse,or other practitioner; or a spouse or guardian, such as a parent. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose.

The subject can be monitored for reactions to the treatment, such asedema or hemorrhaging. For example, the patient can be monitored by MRI,such as daily or weekly following injection, and at periodic timeintervals following injection.

The subject can also be monitored for an improvement or stabilization ofdisease symptoms. Such monitoring can be achieved, for example, byserial clinical assessments (e.g., using the United Parkinson's DiseaseRating Scale) or functional neuroimaging. Monitoring can also includeserial quantitative measures of striatal dopaminergic function (e.g.,fluorodopa and positron emission tomography) comparing treated subjectsto normative data collected from untreated subjects. Additional outcomemeasures can include survival and survival free of palliative therapyand nursing home placement. Statistically significant differences inthese measurements and outcomes for treated and untreated subjects isevidence of the efficacy of the treatment.

A pharmaceutical composition containing an anti-SNCA iRNA agent can beadministered to any patient diagnosed as having or at risk fordeveloping a neurodegenerative disorder, such as a synucleinopathy. Inone embodiment, the patient is diagnosed as having a neurodegenerativeorder, and the patient is otherwise in general good health. For example,the patient is not terminally ill, and the patient is likely to live atleast 2, 3, 5, or 10 years or longer following diagnosis. The patientcan be treated immediately following diagnosis, or treatment can bedelayed until the patient is experiencing more debilitating symptoms,such as motor fluctuations and dyskinesis in PD patients. In anotherembodiment, the patient has not reached an advanced stage of thedisease, e.g., the patient has not reached Hoehn and Yahr stage 5 of PD(Hoehn and Yahr, Neurology 17:427-442, 1967). In another embodiment, thepatient is not terminally ill. In general, an anti-SNCA iRNA agent canbe administered by any suitable method. As used herein, topical deliverycan refer to the direct application of an iRNA agent to any surface ofthe body, including the eye, a mucous membrane, surfaces of a bodycavity, or to any internal surface. Formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, sprays, and liquids. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable. Topical administration can also be used as ameans to selectively deliver the iRNA agent to the epidermis or dermisof a subject, or to specific strata thereof, or to an underlying tissue.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An anti-SNCA iRNA agent can be administered to a subject by pulmonarydelivery. Pulmonary delivery compositions can be delivered by inhalationby the patient of a dispersion so that the composition, preferably iRNA,within the dispersion can reach the lung where it can be readilyabsorbed through the alveolar region directly into blood circulation.Pulmonary delivery can be effective both for systemic delivery and forlocalized delivery to treat diseases of the lungs. In one embodiment, ananti-SNCA iRNA agent administered by pulmonary delivery has beenmodified such that it is capable of traversing the blood brain barrier.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

An anti-SNCA iRNA agent can be administered by an oral and nasaldelivery. For example, drugs administered through these membranes have arapid onset of action, provide therapeutic plasma levels, avoid firstpass effect of hepatic metabolism, and avoid exposure of the drug to thehostile gastrointestinal (GI) environment. Additional advantages includeeasy access to the membrane sites so that the drug can be applied,localized and removed easily. In one embodiment, an anti-SNCA iRNA agentadministered by oral or nasal delivery has been modified to be capableof traversing the blood-brain barrier.

In one embodiment, unit doses or measured doses of a composition thatinclude iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include a pump, such as an osmotic pump and,optionally, associated electronics.

An iRNA agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

Dosage. An anti-SCNA iRNA agent can be administered at a unit dose lessthan about 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg perkg of bodyweight, and less than 200 nmole of RNA agent (e.g., about4.4×1016 copies) per kg of bodyweight, or less than 1500, 750, 300, 150,75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075,0.00015 nmole of RNA agent per kg of bodyweight. The unit dose, forexample, can be administered by injection (e.g., intravenous orintramuscular, intrathecally, or directly into the brain), an inhaleddose, or a topical application. Particularly preferred dosages are lessthan 2, 1, or 0.1 mg/kg of body weight.

Delivery of an iRNA agent directly to an organ (e.g., directly to thebrain) can be at a dosage on the order of about 0.00001 mg to about 3 mgper organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.

The dosage can be an amount effective to treat or prevent a disease ordisorder, e.g., a disease or disorder associated with synucleinopathies.

In one embodiment, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject hasPD and the modality is a therapeutic agent other than an iRNA agent,e.g., other than a double-stranded iRNA agent, or sRNA agent. Thetherapeutic modality can be, for example, levadopa or depronil.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an sRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g.,10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. Themaintenance doses are preferably administered no more than once every 5,10, or 30 days. Further, the treatment regimen may last for a period oftime which will vary depending upon the nature of the particulardisease, its severity and the overall condition of the patient. Inpreferred embodiments the dosage may be delivered no more than once perday, e.g., no more than once per 24, 36, 48, or more hours, e.g., nomore than once every 5 or 8 days. Following treatment, the patient canbe monitored for changes in his condition and for alleviation of thesymptoms of the disease state. The dosage of the compound may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disease state is observed, if the disease state hasbeen ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. In another embodiment, the iRNA agentspecies has sequences that are non-overlapping and non-adjacent toanother species with respect to a naturally occurring target sequence.In another embodiment, the plurality of iRNA agent species is specificfor different naturally occurring target genes. In another embodiment,the iRNA agent is allele specific.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, orpulmonary. For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) can include a single treatment or,preferably, can include a series of treatments. It will also beappreciated that the effective dosage of an iRNA agent such as an sRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays as described herein. For example,the subject can be monitored after administering an iRNA agentcomposition. Based on information from the monitoring, an additionalamount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA, e.g., an SNCA RNA. The transgenic animal can bedeficient for the corresponding endogenous RNA. In another embodiment,the composition for testing includes an iRNA agent that iscomplementary, at least in an internal region, to a sequence that isconserved between the target RNA in the animal model and the target RNAin a human.

Kits. In certain other aspects, the invention provides kits that includea suitable container containing a pharmaceutical formulation of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). In certain embodimentsthe individual components of the pharmaceutical formulation may beprovided in one container. Alternatively, it may be desirable to providethe components of the pharmaceutical formulation separately in two ormore containers, e.g., one container for an iRNA agent preparation, andat least another for a carrier compound. The kit may be packaged in anumber of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Design of iRNA Agents Targeting SNCA

Double stranded RNAs having the sequences described in Table 1 weresynthesized. FIG. 1A shows the sequence of the full-length SNCA mRNA,and the target sites of the dsRNAs SNCA1-9 (Table 1).

The sequences of SNCA6, 7, 8, and 9 were designed using the dsRNASelection Tool developed at the Whitehead Institute (Cambridge, Mass.)and available free on-line. By using the dsRNA Selection Tool, allpossible siRNAs having a GC content between 30 and 70% were selected,except (a) any sequences containing runs of four or more A, T or Gresidues, and (b) any sequences with more than seven consecutive GCpairs in a row. A total of 237 candidate siRNAs matched this criteria.The 237 candidate siRNAs were then screened against the human UniGenedatabase (Pontius et al., UniGene: a unified view of the transcriptome.In: The NCBI Handbook. Bethesda (Md.): National Center for BiotechnologyInformation; 2003) to identify those siRNAs having a sequence that onlymatched human alpha-synuclein. The screening was performed using BLASTsearch technology (Altschul et al., Nucleic Acids Res. 25:3389-3402,1997). The identified subset of siRNAs was screened against mouseUniGene using BLAST to identify those siRNAs having a sequence that onlymatches the alpha-synuclein gene in mouse. Thirteen sequences wereidentified, and four non-overlapping duplexes (SNCA6, 7, 8, and 9) wereselected for use in the assays described below.

TABLE 1 dsRNA sequences SEQ ID dSRNA^(a) NO Strand Sequence^(b) SNCA1 3Sense 5′-GGUGUGGCAACAGUGGCUGAG-3′ 4 Antisense3′-UACCACACCGUUGUCACCGACUC-5′ SNCA2 5 Sense 5′-AACAGUGGCUGAGAAGACCAA-3′6 Antisense 3′-CGUUGUCACCGACUCUUCUGGUU-5′ SNCA3 7 Sense5′-AUUGCAGCAGCCACUGGCUUU-3′ 8 Antisense 3′-CGUAACGUCGUCGGUGACCGAAA-5′SNCA4 9 Sense 5′-AAGUGACAAAUGUUGGAGGAG-3′ 10 Antisense3′-CGUUCACUGUUUACAACCUCCAC-5′ SNCA5 11 Sense 5′-GAAGAAGGAGCCCCACAGGAA-3′12 Antisense 3′-UACUUCUUCCUCGGGGUGUCCUU-5′ SNCA6 13 Sense5′-CGGGUGUGACAGCAGUAGCdTdT-3′ 14 Antisense 3′-dTdTGCCCACACUGUCGUCAUCG-5′SNCA7 15 Sense 5′-UCCUGACAAUGAGGCUUAUdTdT-3′ 16 Antisense3′-dTdTAGGACUGUUACUCCGAAUA-5′ SNCA7s 17 Sense5′-U*CCUGACAAUGAGGCUUAUdT*dT-3′ 18 Antisense3′-dT*dTAGGACUGUUACUCCGAAU*A-5′ SNCA8 19 Sense5′-CUACGAACCUGAAGCCUAAdTdT-3′ 20 Antisense 3′-dTdTGAUGCUUGGACUUCGGAUU-5′SNCA 21 Sense 5′-C*UACGAACCUGAAGCCUAAdT*dT-3′ 8s1 22 Antisense3′-dT*dTGAUGCUUGGACUUCGGAU*U-5′ SNCA 23 Sense5′-C*UACGAACCUGAAGCCUAAdT*dT-3′ 8s2 24 Antisense3′-dT*dTGAUGCUUGGACUUCGGAU*U-5′ SNCA9 25 Sense5′-CUAUUGUAGAGUGGUCUAUdTdT-3′ 26 Antisense 3′-dTdTGAUAACAUCUCACCAGAUA-5′ALN- 27 Sense 5′-GAACUGUGUGUGAGAGGUCCU-3′-F DP- 28 Antisense3′-C*C*CUUGACACACACUCUCCAGGA-5′ 3000 SiRNA 29 Sense5′-GACGUAAACGGCCACAAGUUC-3′ Mr 30 Antisense 3′-CGCUGCAUUUGCCGGUGUUCA-5′SNCA 50 Sense 5′-C*UAUGAGCCUGAAGCCUAAdT*dT-3′ 8s2m 51 Antisense3′-dT*dTGAUACUCGGACUUCGGAU*U-5′ ^(a)SNCA name designations areequivalent to Mayo designations (e.g., SNCA 1 is equivalent to Mayo1);^(b)nucleotides marked with * carry a phosphorothioate modification;underlined nucleotides carry a 2′-O-Me modification; “F” indicates afluorescein conjugate

Example 2 SNCA dsRNAs Decreased Protein Expression In vitro

Neuroblastoma cells (BE(2)-M17) were co-transfected with 50 nM dsRNA anda plasmid expressing either EGFP or an α-synuclein-EGFP (EGFP/NACP)fusion protein (as used herein NACP is synonymous with the gene productof SNCA). Expression of the EGFP and EGFP/NACP fusion proteins wasassayed by Western blot analysis (FIG. 2).

The in vitro cell-based assay monitors the ability of the test dsRNAs ofTable 1 to downregulate expression of an SNCA RNA. The SNCA target RNAin these experiments is fused to an EGFP RNA. Antibodies against EGFPfacilitate the detection of an EGFP/NACP fusion protein translated fromthe RNA.

Control experiments used in this assay included the use of a dsRNAtargeting a luciferase RNA (see the lanes marked “siRNA Mr” in FIG. 2),and cells not transfected with siRNA. Antibodies against alpha tubulinwere used as controls to monitor the amount of total protein loaded ineach lane. The control experiments indicated that EGFP and the EGFP/NACPproteins are expressed in about equal amounts when in the absence ofanti-SNCA dsRNA. The strongest down-regulatory effect of EGFP/NACPexpression was observed with Mayo2, Mayo7, and Mayo8 dsRNAs (as usedherein, MayoX siRNAs are synonymous with SNCAX dsRNAs). A weaker effectwas observed with Mayo1, Mayo6, and Mayo9 dsRNAs. siRNA Mr affectedexpression of both the vector derived EGFP and the EGFP/NACP conjugate,demonstrating the suitability of the assay.

The inhibitory effect of the most effective dsRNAs (Mayo2, Mayo7, andMayo8) was examined at varying dsRNA concentrations during a 24 hincubation (FIG. 3). The IC₅₀ value was determined to be less than 1 nM.

The inhibitory effect of the most effective siRNAs was tested inslowly-dividing neuroblastoma cells in cultures with low levels ofserum. BE(2)-M17 cells were transfected with a plasmid expressing theEGFP/NACP fusion protein alone (control) or cotransfected with thedsRNAs Mayo2, Mayo7, or Mayo8. Expression of the EGFP/NACP protein wasmonitored over a period of six days. Fusion protein expression wasobserved to be effectively silenced for at least three days (FIG. 4).The dsRNAs also inhibited endogenous protein expression in similar cellsfor at least three days (FIG. 5). The Mayo2, Mayo7, and Mayo8 siRNAsalso inhibited expression of endogenous alpha synuclein RNA in slowlydividing cells (FIG. 6). After 6 days, Mayo2 and Mayo8 continued toeffectively inhibit endogenous SNCA expression. Levels of alphasynuclein mRNA in the cells were measured by the Taqman® method ofquantitative RT-PCR normalized against 18S rRNA expression levels.Mayo9, which targets the 3′UTR of SNCA, did not inhibit expression ofendogenous alpha synuclein RNA.

The efficacy of the Mayo2, 7, and 8 dsRNAs were tested against mouseSNCA. Cells were cotransfected with the dsRNAs and a plasmid encodingEGFP alone (vector) or EGFP-NACP of human or mouse origin. Expression ofEGFP and EGFP-NACP was assayed by Western blot. While all three dsRNAsinhibited expression of human EGFP-NACP, only Mayo2 inhibited expressionin mouse EGFP-NACP (FIG. 7). The human and mouse mRNA sequence isidentical at the Mayo2 locus, but diverges by two nucleotides at each ofthe Mayo7 and Mayo8 loci.

SNCB (beta-synuclein) shares sequence similarity with alpha-synuclein atthe Mayo2 locus, but differs in sequence by four nucleotides. Theefficacy of the Mayo2 was tested against SNCB. BE(2)-M17 cells weretransfected with a plasmid expressing the dsRNAs Mayo2 or Mayo9.Expression of endogenous SNCA and SNCB RNA was assayed by Taqman® methodquantitative RT-PCR. Mayo2 inhibited expression of SNCA but notexpression of SNCB (FIG. 11). As was expected, Mayo9 did not inhibitexpression of SNCB or SNCA.

Example 3 Stability of SNCA siRNAs

The stability of the sense and antisense strands of the SNCA siRNAs wasexamined in 90% mouse serum or 90% human serum, and in mouse braintissue. To perform the stability assays, siRNA was radioactively labeledon the sense or antisense strand (both strands were assayed forstability in the serum and brain tissue). Protein extracts were preparedfrom mouse brain, and 100 nM siRNA duplex was incubated with the extractat 37° C. At time points over the course of 4-5 hours, sample wasremoved and analyzed on a polyacrylamide denaturing gel.

The stability of Mayo2, 7, and 8 was tested in mouse serum and brainextract. Further, the cleavage sites of Mayo7 and Mayo8 were mapped byT1 analysis (FIGS. 8A and 8B). RNAse T1 cleaves 3′ of G nucleotides, andT1 digestion of an RNA that has a known sequence provides orientationand a basis for comparison to detect non-RNAse T1 cleavage sites. T1 wasused to map the cleavage sites of Mayo7 (also called SNCA7, orAL-DUP-1477) and Mayo8 (also called SNCA8, or AL-DUP-1478) siRNAs (FIGS.8A and 8B, respectively, and Table 1). Mayo7 and 8 were 5′ end labeledwith ³²P on the sense strand, and RNAse T1 digestion was performed forfour hours. The samples were analyzed by electrophoresis. Mayo7 wasfound to be susceptible to endonucleolytic cleavage 3′ of U16 and U17.Mayo8 was found to be susceptible to endonucleolytic cleavage 3′ of U16.

To increase stability of the Mayo7 and Mayo8 siRNAs, nucleotides weremodified with a 2′-O-Me group or a phosphorothioate linkage to createMayo7s, Mayo8s1, and Mayo8s2 (Table 1). The modified siRNAs (50 nM) werecotransfected with an EGFP-NACP vector into cells as described above.Untransfected cells served as a control. Gene expression was monitoredby Western blot analysis. Each of the three modified siRNAs inhibitedexpression of the EGFP-NACP construct (FIGS. 9A and 9B).

The modified and unmodified Mayo8 siRNAs were analyzed by Stains-All(cat. #E9379, Sigma, St. Louis, Mo.), which was performed as follows.All solutions were prepared in nuclease-free water (cat. #9930, Ambion,Austin, Tex.), using nuclease-free reagents. A 50 μM stock of dsRNA foruse in the stability assays was prepared by mixing 50 μM sense strandRNA and 50 μM antisense strand in 1×PBS. This mixture was incubated at90° C. for 2 minutes to denature the nucleic acids, then 37° C. for onehour for annealing.

To perform the stability assay, human serum from clotted male wholeblood type AB (cat. #H1513, Sigma, St. Louis, Mo.) was used. Serum wasthawed on ice, and mixed with dsRNA to a final concentration of about4.5 μM (i.e., about 4.2 μg, or about 300 pmoles dsRNA). At time point“0,” one control sample was frozen on dry ice immediately followingaddition of dsRNA to serum, and the sample was stored at −80° C. Forother time points (15, 30, 60, 120, and 240 minutes in human serum), thesamples were incubated at 37° C. in a Thermomixer (Eppendorf, Hamburg,Germany). At each endpoint, the samples were frozen on dry-ice andstored at −80° C.

To extract the RNA from the serum, samples were thawed on ice, and then0.5 M NaCl (nuclease free; cat#9760, Ambion, Austin, Tex.) was added tothe sample to yield a final concentration of about 0.45 M NaCl. Thesample was vortexed briefly (about 5 seconds), and then transferred to aprepared and chilled Phase Lock-Gel-Eppis (Eppendorf, Hamburg, Germany).Five hundred microliters phenol:chloroform:isoamyl alcohol (25:24:1) and300 μL chloroform were added to the mix. The sample was vortexed brieflyfor 30 seconds, then centrifuged at 13,200 rpm for 15 minutes at 4° C.

The aqueous phase was transferred to a clean eppendorf tube, and 3MNaOAc, pH 5.2, was added to a final concentration of about 0.1M NaOAc.The solution was vortexed for about 20 seconds and then 1 μL of GlycoBlue (Ambion, Austin, Tex.) was added. The solution was vortexed brieflyand gently, then 1 mL ice-cold 100% ethanol was added. The solution wasvortexed for about 20 seconds, then stored at −80° C. for one hour, orat −20° C. overnight to precipitate the RNA. Following precipitation,the mixture was centrifuged at 13,200 rpm for 30 min. at 4° C., and theRNA pellet was washed with 500 μL 70% ethanol. The pellet was air-dried,then 30 μL of gel loading buffer (95% formamide, 50 mM EDTA,Xylenecyanol, bromophenol blue) was added to the mix, and the mixvortexed for 2 minutes to resuspend.

The RNA sample was analyzed on a 20 cm×20 cm×0.8 mm(length×width×thickness) 20% polyacrylamide gel. To make the gel, 24 g 8M Urea, 25 mL 40% (19:1) Acrylamide, and 8 mL formamide was mixed in1×TBE in a 50 mL solution. Polymerization was activated by 50 uL Temedand 200 uL 10% APS (ammonium persulfate). The gel was run in 1×TBE. Thegel was pre-run for 30 minutes at 40 mA. The samples were heated at 100°C. for 5 min. and then immediately chilled on ice. For controlexperiments, 2 μL of dsRNA was mixed with 8 μL of gel loading buffer.The samples were centrifuged at 13,200 rpm (20 seconds, 4° C.) and 10 μLwas loaded onto the gel. The gel was run for about one hour at 40 mA.

To visualize the RNA, the gel was stained with Stains-All solution (cat.#E9379, Sigma, St. Louis, Mo.) (100 mg Stains-All dissolved in 800 mLformamide:water (1:1 v/v)) for 30 minutes. The gel was destained inwater for 30-60 minutes as needed. The gel was them imaged on a scannerand analyzed.

The results of the stability assay are shown in FIGS. 10A, 10B and 10C.Comparison indicates that the unmodified SNCA8 dsRNA is rapidlydegraded, the partially modified dsRNA (SNCA8s1) is partiallystabilized, and the further modified SNCA8s2) is the most stable of thethree duplexes.

Example 4 In vivo Analysis of siRNA Biodistribution

To determine whether siRNA could be delivered into neural cells in vivo,siRNA targeting luciferase (ALN-DP-3000) (Table 1) was conjugated with afluorescein label and administered to distinct areas of mouse brain bystereotactic injection (Table 2). ALN-DP-3000 was injected into thecortex, the hippocampus, and the globus pallidus areas of the brain. Atdifferent time points post-injection, brain tissue was harvested,sectioned, and examined microscopically for the localization of thefluorescently-labeled siRNA. In all brain regions examined (cortex,hippocampus, and globus pallidus), and at all time pointspost-injection, siRNA was found to localize to extracellular spaces aswell as intracellularly.

TABLE 2 Analysis of ALN-DP-3000 biodistribution in brain tissue Timepost- Stereotactic injection of Injection Site coordinates tissueanalysis Cortex AP −0.0  1 hr. L −3.5 DV −1.8 Cortex AP −0.0 24 hr. L−3.5 DV −1.8 Hippocampus AP −1.8  1 hr. L −2.2 DV −1.2 Hippocampus AP−1.8 24 hr. L −2.2 DV −1.2 Globus Pallidus AP −0.3  1 hr. L −1.8 DV −3.5Globus Pallidus AP −0.3  2 hr. L −1.8 DV −3.5 Globus Pallidus AP −0.3  4hr. L −1.8 DV −3.5

To assess activity in vivo, siRNA duplexes were administered bystereotactic injection to the substantia nigra of wild-type mice(C57BL/6 mice; Taconic, Germantown, N.Y.). Coordinates for stereotacticinjection were as follows: AP −3.4 mm; L −1.5 mm; DV −3.8 mm. Threeanimals each received two microliters of a 200 μM solution of Mayo-8s2msiRNA in phosphate buffered saline (PBS) (Table 1; FIG. 12A, barslabeled “E”). As a control, three animals were injected with PBS (FIG.12B, bars labeled “C”). Twenty-four hours after injection, animals weresacrificed and brains were removed. Tissue blocks encompassing theinjection track were dissected and total RNA was isolated from about 100mg of tissue using Trizol reagent (Invitrogen, Carlsbad, Calif.).Taqman® quantitative RT-PCR (Applied Biosystems, Foster City, Calif.)was used to measure relative levels of alpha-synuclein mRNA. As anormalization standard, 18S rRNA was measured separately (“individualtube”) or in the same Taqman®reaction with alpha-synuclein (“duplex”).Reverse transcription was performed at 48° C. for 30 minutes, and PCRwas performed for 40 cycles of (95° C. for 15 sec., 65° C. for 1 min.).Each experiment was performed three times, and in each experiment,reactions were performed in quadruplicate. The comparative count method(ΔΔCt) was used to determine relative levels of alpha synuclein comparedto control (Heid et al., Genome Res. 6:986-994, 1996). SDS 2.1 software(Applied Biosystems, Foster City, Calif.) was applied with automaticthreshold values and automatic outlier removal.

The preliminary results shown in FIGS. 12A and 12B indicated that, onaverage, the Mayo-8s2m siRNA specifically decreased SNCA mRNA levels inmouse brain, as relative levels of the control 18S rRNA were notaffected.

Example 5 Silencing of Endogenous Alpha-synuclein by IntraparenchymalInfusion of siRNA

Methods

Using stereotactic surgery, infusion cannulae were implanted into thehippocampus of eight-week old, female B6 mice (coordinates from bregma:x=(−)2.0, y=(−)1.5, z=2.0 calculated from Paxinos and Franklin, TheMouse Brain in Stereotaxic Coordinates). Cannulae were implanted intothe right hemisphere of the brain. The cannulae were connected viacatheters to osmotic mini-pumps (Alzet model 1002) containingapproximately one hundred microliters of 2.1 mM siRNA solution inPhosphate Buffered Saline (PBS). The pumps were implantedsubcutaneously. The infusion rate of 0.25 microliters per hour resultedin a dose of approximately 180 micrograms of siRNA per day. Infusioncontinued for a period of fifteen days. Treatment groups were: PBS(n=10), alpha-synuclein duplex (SNCA siRNA; n=8), cholesterol conjugatedalpha-synuclein duplex (SNCA siRNA-chol; n=8), luciferase control duplex(n=8), cholesterol conjugated luciferase control duplex (n=10). Thesequences of the duplexes, as well as chemical modifications are shownbelow in Table 3.

TABLE 3 siRNA Sequence Luc control S 5′ cuuAcGcuGAGuAcuucGATsT 3′ (SEQID NO: 52) AS 5′ UCGAAGuACUcAGCGuAAGTsT 3′ (SEQ ID NO: 53) Luc control S5′ cuuAcGcuGAGuAcuucGATsTs-chol 3′ (SEQ ID NO: 54) chol AS5′ UCGAAGuACUcAGCGuAAGTsT 3′ (SEQ ID NO: 53) SNCA S5′ CsuAUGAGCCUGAAGCcuaATsT 3′ (SEQ ID NO: 55) AS5′ usuAGGCUUCAGGCUCAuAGTsT 3′ (SEQ ID NO: 56) SNCA chol S5′ CsuAUGAGCCUGAAGCcuaATsT-chol 3′ (SEQ ID NO: 57) AS5′ usuAGGCUUCAGGCUCAuAGTsT 3′ (SEQ ID NO: 56) Key A, C, G,U-ribonucleotides c, u-2′-OMe nucleotides s-phosphorothioate linkageT-thymidine

Following the infusion period, brains were collected and the regionscorresponding to the hippocampus were dissected from each hemisphere.Total RNA was isolated and used to prepare cDNA by random hexamerpriming. Relative levels of alpha-synuclein were measured by TaqMan®quantitative PCR using gene expression MGB probes (SNCA Mm0044733_ml,GAPDH Mm99999915_gl, HPRT Mm00446968_ml, Tau Mm00521988_ml; AppliedBiosystems). For more accurate normalization among tissues, levels ofGAPDH, HPRT and tau were measured and used to determine a normalizationfactor. Relative levels of alpha-synuclein were calculated for the rightand left hemispheres from each animal, and group means and standarddeviations were calculated.

Results

A decrease of alpha-synuclein expression of approximately 30% (right vsleft side) was measured in the animals infused with the SCNA siRNA.Statistical significance (p=0.036) was determined by T-test (FIG. 13).

Example 7 In situ Hybridization Showing Silencing of Endogenousα-synuclein by Intraparenchymal Infusion of siRNA

Infusion cannulae were implanted into the hippocampus of eight-week old,female B6 mice (coordinates from bregma: x=(−)2.0, y=(−)1.5, z=2.0calculated from Paxinos and Franklin, The Mouse Brain in StereotaxicCoordinates). The cannulae were connected via catheters to osmoticmini-pumps (Alzet model 1002) containing approximately one hundredmicroliters of 2.1 mM siRNA solution in Phosphate Buffered Saline (PBS).The pumps were implanted subcutaneously. The infusion rate of 0.25microliters per hour resulted in a dose of approximately 180 microgramsof siRNA per day. Infusion continued for a period of fifteen days.Treatment groups were: PBS (n=10), alpha-synuclein duplex (SNCA siRNA;n=9), luciferase control duplex (n=10). The sequences of the duplexes,as well as chemical modifications are shown below (Table 4).

TABLE 4 siRNA Sequence Luc control S 5′ cuuAcGcuGAGuAcuucGATsT 3′ (SEQID NO: 52) AS 5′ UCGAAGuACUcAGCGuAAGTsT 3′ (SEQ ID NO: 53) SNCA S5′ CsuAUGAGCCUGAAGCcuaATsT 3′ (SEQ ID NO: 55) AS5′ usuAGGCUUCAGGCUCAuAGTsT 3′ (SEQ ID NO: 56) Key A, C, G,U-ribonucleotides c, u-2′-OMe nucleotides s-phosphorothioate linkageT-thymidine

Following the infusion period, brains were dissected rapidly. To ensuresampling consistency, the brain was placed in a tissue matrix and theregion anterior and posterior to the hippocampus was removed using aflat blade. The resulting three brain segments were snap frozen on dryice and stored at −80° C. until use. Frozen sections were cut at 15 μmon a cryostat at −18° C. throughout the entire hippocampus and air driedfor 20 minutes before freezing at −80° C. On the day of the experiment,frozen sections were removed on dry ice and dried quickly on a slidewarmer at 55° C., then fixed in 4% paraformaldehyde in 0.1M Sorensen'sPhosphate buffer for 20 minutes, washed twice in PBS and then dehydratedin ascending alcohols. Hybridization was then performed at 37° C.overnight, in a moist chamber, with approximately 0.02 ng of [α−³³P]dATP 3′ end labeled probe per 1 μl of hybridization buffer (4×SSC, 1×Denhardt's solution, 50% (w/v) de-ionised formamide, 10% (w/v) dextransulphate, 200 mg/μl herring sperm DNA). The probe(5′GGTCTTCTCAGCCACTGTTGTCACTCCATGAACCAC′3) (SEQ ID NO: 58) was designedto exon 3 on mouse SNCA. Specific activity of the probe was >1×10⁸cpm/μg and after dilution in hybridization buffer corresponded to ˜1×10⁴cpm/μl. Control hybridizations were also set up that contained a 50-foldmolar excess of unlabelled probe to determine non-specific signal.Slides were washed in 1×SSC at room temperature (RT) to remove excesshybridization buffer; three times, each for 30 minutes, at 55° and at RTfor 60 minutes. Slides were then dipped for 30 seconds in 70% (v/v)ethanol/300 mM ammonium acetate, then for 30 seconds in absolutealcohol, air dried and co-exposed with ¹⁴C microscale standards(Amersham) to Biomax MS film (Kodak) for 7-10 days.

The Metamorph software (Universal imaging) was used to performdensitometry. Specifically, optical density of mRNA labeled with theSNCA specific probe was measured in a standard square with and area of240 pixels² in the cortex. Optical density was measured and values werecompared to optical density of the known ¹⁴C standards. From thesevalues and a graph was constructed and concentration of radioactivity innCi/g in each sample was extrapolated. A t-test was used to determine ifthere was difference between groups.

Results

There was a reduction in the expression of siRNA in the hippocampus andcortex in the injected side (right) compared uninjected side (left) ofthe siRNA treated animal (B). The PBS animal did not show a reduction inthe injected side (A) in all the animals used in the in situhybridization experiment (n=3). Densitometry analysis of the in situhybridization film showed significant reduction (˜60%) in SNCA mRNAexpression in the injected side compared to the uninjected side (C) ofthe cortex (*p=0.003, t-test). Boxes in A and B show cortical regionmeasured (FIG. 14).

Example 7 Method of Treating a Patient Diagnosed with a Synucleinopathy

A patient diagnosed with a synucleinopathy can be administered apharmaceutical composition containing an iRNA agent that targets theSCNA gene. The composition can be delivered directly to the brain by adevice that includes an osmotic pump and mini-cannula and is bilaterallyimplanted into the patient.

Prior to implantation of the device, the patient receives an MRI withstereotactic frame. A computer-guided trajectory is used for delivery ofthe cannula to the brain. The mini-pump device is implanted into theabdomen, and then the patient is hospitalized for 2-3 days to monitorfor hemorrhaging.

Approximately two weeks post-implantation of the pump, the patient canreceive an MRI to check the implanted device. If the human is healingwell, and no complications have occurred as a result of implanting thedevice, then the anti-SNCA composition can be infused into the pump, andinto the cannula. A test dose of the anti-SNCA agent can be administeredprior to the initiation of the therapeutic regimen.

MRIs taken at 3 months, six months, and one year following the initialtreatment can be used to monitor the condition of the device, and thereaction of the patient to the device and treatment with the iRNA agent.Clinicians should watch for the development of edema and an inflammatoryresponse. Following the one-year anniversary of the initiation of thetreatment, MRIs can be performed as needed.

The patient can be monitored for an improvement or stabilization indisease symptoms throughout the course of the therapy. Monitoring caninclude serial clinical assessments and functional neuroimaging, e.g.,by MRI.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of inhibiting alpha-synuclein (SNCA) expression in a mammalcomprising administering an iRNA agent directly to the brain of saidmammal, wherein the iRNA agent comprises an antisense strand comprisingSEQ ID NO: 22 and a sense strand comprising SEQ ID NO: 21, wherein eachstrand is 21-25 nucleotides in length.
 2. The method of claim 1, furthercomprising a step of identifying a synucleinopathy in said mammal priorto the administering of said iRNA agent.
 3. The method of claim 1,wherein said SNCA expression is reduced by at least 50% relative toexpression in cells that had not been contacted by said iRNA agent. 4.The method of claim 2, wherein said mammal is a human.
 5. The method ofclaim 1, wherein said iRNA agent is administered by stereotacticinjection into the brain of said mammal.
 6. The method of claim 1,wherein said iRNA agent is administered by intraparenchymal infusion orinjection.
 7. The method of claim 4, wherein said iRNA agent isadministered by stereotactic injection into the brain of said human. 8.The method of claim 4, wherein said iRNA agent is administered byintraparenchymal infusion or injection.
 9. The method of claim 1 or 4,further comprising a step of measuring SNCA expression in said mammal.10. The method of claim 1 or 4, wherein said SNCA expression is measuredbefore and after said administration of said iRNA agent.
 11. The methodof claim 1, wherein each strand of the iRNA agent is 21 nucleotides inlength.